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TGGE Manual

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1. 10 20 30 40 50 60 70 80 90 100 1i0 120 130 140 Stack index Sequence TGGE System 70 8 Optimizing parallel TGGE by perpendicular TGGE 8 1 Check short DNA fragments for their melting behavior All short DNA fragments 100 150 bp should be checked first by a perpendicular TGGE gel This is not only a good place to start for practical optimization of parallel TGGE but also verifies the reversible melting behavior of the DNA fragment fig 23 Reversible melting can only occur if the DNA fragment consists of at least two separate melting domains fig 23 a c d Reversible melting behavior must be verified since it is required for successful parallel TGGE analysis Thus be sure to check all fragments on a perpendicular TGGE gel which do not contain 40 bp GC clamp or which have not been evaluated with the aid of the POLAND program Short DNA fragments without GC clamp Y lt n single strands single stands a Reversible melting pattern b Irreversible melting pattern GC clamp is required GC clamped fragments _ single strands lt unge strands ec Reversible melting pattern d Reversible melting pattern Figure 23 Short DNA fragments without GC clamp TGGE System 71 8 2 From perpendicular to parallel TGGE Perpendicular TGGE routinely uses a standard temperature gradient from 20 60 C in combination with buffers that contain minimu
2. ccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeees 6 2 General Recommendations z 5 3 552252 33H RER 8 2 1 Safety wamingsS ae Ride nal tt ad leit Se eS Sih oad 8 PAP NOLES TOR USC ecc25 coef ceases er E deh ea deh oa deh ce oe oe 8 3 Components of the TGGE System unnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn 9 3 1 Electrophoresis unit essen nasange 10 32 Controller uitsere re een e re e 17 3 2 1 Instrument keys and ports 22 2 cence eee ene 17 3 2 2 Programming of the TGGE Conttroller ccccceeeeeeeeeeeeeeeeeeeeeeeeees 18 4 Sample preparation 24 4 1 Purity Ol samples ER ee eigen a 24 4 2 Quantity and volumes of samples 244444444444nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 25 5 Setting up polyacrylamide gels unnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 25 5 1 Selecting Concentration of PAA gels eee 25 9 2 S tting Up Ae Gel SOMMON ees este lee ccs aeieea eeacedelees 25 5 3 Some remarks corresponding to standard TGGE conditions 28 5 4 Assembling the gel sandwich 30 5 5 Disassembling the gel sandwich 32 6 Electrophoresis with the TGGE System ee ji i ines 33 6 1 tEle6troph resis condilions siciiic Sarasin one 33 6 2 Preparing the electrophoresis unit cccccccceeceeeeeeeeeeeeeeeeeeeeesseeeeeeees 33 6 3 Perpendicular TO GE n en 35 BA Parallel TOGE ern ee 39 6 5 Silver Sstalnings
3. Error messages TGGE System check connection to thermoblock warning gradient too large max grad T1 gt T2 45 C A Bno gt L1 C quit D enter or warning gradient too large max grad T1 gt T2 45 C A Bno T1 C quit D enter program no TEST pgm is active A copy B del C quit D display program no __ name not programmed AT BJ Cauit Denter 1 11 __ L6 entry required T2 A BLU Cquit D or 1 11 __ L6 entry required T1 T2 A BT1 Cqut Do TGGE connector cable is not connected to gradient block and or system Controller Programmed temperature gradient too large It is possible to review a program during run After pressing SC program and the corresponding store number a warning message appears This program number has not been programmed No temperature or time has been programmed TGGE System 24 4 Sample preparation 4 1 Purity of samples Due to the high sensitivity of the staining procedure after TGGE it is recommended to use purified DNA RNA or protein samples Any impurities might be misinterpreted after TGGE thereby making the analysis of gels difficult Nevertheless it is possible to use even crude mixtures for TGGE analysis PCR amplified DNA fragments can usually be analyzed without purification But note that the presence of high amounts of nonspecific secondary PCR products may result in difficul
4. References Lerman L S Fischer S G Hurley l Silverstein K amp Lumelsky N 1984 Ann Rev Biophys Bioeng 13 399 423 Fischer S G amp Lerman L S 1982 Proc Natl Acad Sci USA 80 1579 1583 Riesner D Henco K amp Steger G 1991 In Advances in Electrophoresis Vol 4 Chrambach A Dunn M J amp Radola B J eds VCH Verlagsgesellschaft Weinheim pp 169 250 Temperatur gradient gel electrophoresis A method for the analysis of conformational transitions and mutations in nucleic acids and proteins Institut f r Physikalische Biologie Department of Biophysics Heinrich Heine Universit t D sseldorf Germany Feb 26 1999 G Steger M Labensky A J ger TGGE System 65 7 3 5 How to use the new Poland program Poland service request form Sequence title line Sequence plain format no numbers max 1000 nts min 5 nts Mismatched positions comma separated numbers Thermodynamic parameters DNA 100 mM NaCl Klump Oligonucleotide Long double strand an B is function of seq length default R 1 0E 3 M Dissociation constant R C E Strand concentration COS default 1 0E 6 M Low temperature limit IHigh temperature limit Temperature step default 40 0 C default 110 0 C size default 2 0 C Temperature range Tm p 50 3d Mobility Melting Diff melting plot plot plot curve curve Which gr
5. or t click here to etl form to defaults If you have comments or suggestions on this service you can send us mail here oligonucleotide or BiophyswwW G Steger Oct 1996 TGGE System 54 Working with the web based POLAND program only need 4 steps 1 Enter DNA sequence lt 1000 bases 2 Enter mismatch position optional 3 Choose GIF format A Press submit TGGE System 55 7 3 The new Poland program New Server 7 3 1 About the Poland service The Poland server will calculate thermal denaturation profiles and temperature dependent UV absorbance or gel mobility of double stranded RNA DNA or RNA DNA hybrids based on sequence input and parameter settings in the Poland request form Details of the Poland program are given below The program used in these calculations was developed by Gerhard Steger for comparing theoretical predictions to experimental data mainly optical denaturation profiles taken at 260 and 280 nm and TGGE temperature gradient gel electrophoresis experiments The original version was written in VAX Fortran VMS using the Graphics Kernel System GKS for data presentation 7 3 2 Program specific information Calculation is based on D Poland s algorithm including the modification by Fixman amp Freire in the implementation described by Gerhard Steger The Poland algorithm calculates the denaturation profile for double stranded nucleic acid using nearest neighbor stacking i
6. Fragment A Fragment B Fragment C Fragment D Fragment E Fragment F Fragment G Fragment H Fragment Fragment J Fragment K Fragment L Fragment M Fragment N Figure 22 Schematic TempPlot diagram of a DNA sequence TGGE System 68 Fragments which have not been clamped may also be analyzed on TGGE but they must contain at least two melting domains In this case the most stable melting domain may act as a natural GC clamp provided that electrophoresis is terminated before this second domain reaches its respective Tm Under these experimental conditions nucleotide changes within this highest melting domain will not lead to a shift of bands on the TGGE gel and hence will not be detectable In conclusion the absence of GC clamps will still allow nearly 100 detection rate for mutations in the low melting temperature domain s but virtually no ability to detect mutations in the domain with the highest Tm 7 4 1 Asymmetric GC clamps for PCR primers used for TGGE analysis The 5 end or the 3 end of the primer for the end of the segment at which a clamp is optimal must carry a GC clamp The length of the clamp depends on the sequence of the sample The denaturing behavior of the modified sample can be tested using the POLAND software short GC clamp 23 bp cccgce cgcgce cccge cgccc gcc long GC clamp 40 bp cgccc gccgce gcccc gcgcc cggcc cgccg ccccc gcccg long GC camp 39 bp ccccg ccccc gccge c
7. 232H222 2ER 44 6 6 Elhidiim Bromide staining rer ze EEE elchbeen 48 Ofr 0 11 0 10 ea 48 6 8 Autoradiography eeen 48 6 9 Elution of DNA from the TGGE gel eneren 48 7 TGGE in analysis of point mutations in dsDNA unnssnsnnnnnnnnnnnnnnnnnnnnnnnnnnnn 49 Il aedi Theoretical background of a detection rate approximating 100 for point mutations calculations with the POLAND program eee 49 7 22 The old Poland program Old Server cccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 51 7 2 1 About the Poland service 2 2 2 teten 51 7 2 2 Program specific information cccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 51 7 2 3 How to use the old Poland program Standard 44444 4 gt 53 7 3 The new Poland program New Server 55 7 3 1 About the Poland Service 2 2 2 2 222 222 2 22 0 ea ee ee 55 7 3 2 Program specific information ss eseeeeeeeseeeeeeeseeeeeeeeeeeeeeeeeeeees 55 7 3 3 References for Poland Service He 56 7 3 4 HELP for Poland Service cccccccceeceececcecceeeeceeceeceeaececueceeaeeueeneeneaes 56 TGGE System 2 7 3 5 How to use the new Poland program cccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 65 7 4 The optimized DNA fragment nnee 66 7 4 1 Asymmetric GC clamps for PCR primers used for TGGE analysis 68 7 4 2 Chemical clamp with P
8. Calculation is based on Poland s algorithm including the modifications proposed by Fixman amp Freire With the original algorithm of Poland computing time is proportional to the square of the sequence length With the modification according to Fixman amp Freire computing time is proportional to 10 times the sequence length but it works only with loop parameters according to Poland 7 3 4 6 SUGGESTIONS Hints for combination of parameters and their values Additional information available RNA DNA RNA DNA lonic_strength_dependence RNA Thermodynamic values according to Turner et al are ideally suited for calculation in 1 M ionic strength after correction of all DeltaS values by 1 021 and all DeltaSGC values by 0 961 These corrections are equivalent to a shift in Tm values of A U stacks by 7 K or 2 respectively and of G C G C stacks by 7 K or 2 respectively Optimal parameter combination for Turner et al d 1 021 1 000 0 961 DeltaS DeltaS A U and DeltaS G C factor n 1 e 3 Dissociation constant B c 1 e 6 R cO 1E 8 to 1E 10 s 1 000 loop parameter Sigma lg internal loops according to Gralla amp Crothers t 90 120 0 5 Temperature range and steps TGGE System 59 Optimal parameter combination for P rschke et al d 1 000 1 040 0 970 DeltaS DeltaS A U and DeltaS G C factor n 1 e 3 Dissociation constant B c 1 e 6 cO 1E 9 to 1E 11 s 1 e 6 loop paramete
9. Pressing SC program during block run Program no 4 Pgm is active A copy B del C quit D display program no 4 name oo A copy B C quit D display In the main menu pressing key B block offers the possibility to start a program After entering a program number or choosing a program from the list SA list the block starts to establish the gradient The timer for the ramping starts immediately The electrophoresis is started as soon as the gradient block reaches the programmed temperature gradient The limiting factor const V mA or W is indicated by an blinking arrow After establishing the gradient line two of the display changes The timer now starts again and counts the electrophoresis running time Additionally the volt hour integrator starts to count It is possible to review a program during run After pressing SC program and the corresponding store number a warning message appears Then it is possible to display the active program D display or to copy it into a new store SA copy It s not possible to change the currently running program By pressing SC quit the main menu appears again Stop block function and electrophoresis program 0 TEST pause stop A B pause C quit D stop L1 25 0 C L6 60 0 C hold 1 pause 0 00 Vh El 250 V 8mA 20W A B Elpho C programs D program 0 TEST continue stop A B contin
10. The electrophoresis conditions depend on the kind of material to be separated e g fragment size differences kind of application e g parallel or perpendicular TGGE sample preparation e g high salt or low salt preparation buffer system Any recommendations can only be used as qguidelines to start with Further improvements to the analysis is easily possible by adjusting the run conditions to the individual needs Voltage 100 V 400 V usually 250 V Current 5mA 25mA usually 10 20 mA Run Time 10 min 2h usually 30 min 6 2 Preparing the electrophoresis unit e Use the leveling eye on the electrophoresis unit and the 4 leveling feet to adjust the unit e Remove the safety lid and fill in max 250 ml of the desired running buffer per buffer chamber e g 0 1 x conc TBE see Appendix 13 2 The same running buffer should only be used once e Soak the pre cut electrode wicks order number 024 020 in the running buffer before use e Drop 0 3 0 5 ml of thermal coupling solution like 0 1 Triton or 0 1 Tween 20 on the surface of the gradient block see figure 13 left panel The thermal coupling solution will increase the adhesion of the Polybond film with the attached polyacrylamide gel and therefore supports temperature equilibration between gradient block and polyacrylamide gel The whole block must be covered by the thermal coupling solution layer No air bubbles must form Figure 13 Positioning of polyacrylami
11. The electrophoresis unit consists of 4 parts 1 safety lid with 2 electric plugs anode and cathode 2 removable electrophoresis chambers each with platinum wires and elec tric connectors volume max 250 ml housing with Peltier element powered gradient block 37 pin connecting cable to control unit Figure 6 Parts of the electrophoresis unit TGGE System 11 T by ty by ty bs by Te EO J L J O O m 2 BEEBE BEE Ti z k u i A eT T aT o O O r fe N Figure 6b Gradient block with temperature lines and marks for positioning the gel slots top Safety lid with arrow indicating the running direction of nucleic acids middle Gradient block covered with safety lid setup for a perpendicular TGGE run bottom left setup for a parallel TGGE run bottom right TGGE System 12 The gradient block is centrally positioned in the middle of the electrophoresis unit and protected by a layer of white foil This foil is necessary to protect the electronic parts beneath it from liquid buffer or other harmful chemicals If the protection foil has been scratched during use stop working and exchange the protection foil with a new one The two opposite sides of the gradient block are marked with lettering T and To Beneath these symbols the Peltier elements whi
12. The glass plate needs 4 Treat glass plate with spacer with treatment with Acryl Acryl Glide Glide Air bubbles between slots The glass plate needs 1 Treat glass plate with spacer with treatment with Acryl Acryl Glide before each use Glide Clean spacers with EtOH Slots are distorted Gel sticks to the slot 1 Treat glass plate with spacer with former and or glass Acryl Glide before each use plate Polybond film with gel 2 Remove slowly the Polybond film have been removed beginning from the bottom or to quick with gel from the glass plate with spacer beginning from the top TGGE System 82 Problem Cause Solutions Electrophoresis unit and Controller Scratches in the white cover Cover film ofthe 1 Remove the cover film and film of the gradient block gradient block damaged replace by a new one Gel running No or minimal current Safety lid is not seated 1 Position the safety lid correctly lt 5 mA Marker dyes stop in properly the gel Assembly of the TGGE 2 Check the assembly of the TGGE system is incorrect system and check plug connections Gel is drying out White 3 Carefully protect the gel against Opaque areas can be evaporation Take special care of seen inside the gel the slots After the sample has migrated into the gel cover the gel with cover film and additionally with the special Cover glass plate with 2 silicone barriers
13. eKO N i e Wait at the electrophoresis chamber until the samples have fully entered the polyacrylamide gel unlike with the former TGGE System of QIAGEN this process will only take 1 3 minutes and have moved about 3 5 mm in the gel e Stop the electrophoresis run open the safety lid e Rinse the now empty slots with 0 5 1 ml running buffer e Cover the polyacrylamide gel including the slots with the 7 x 6 cm pre cut cover film see figure 17 A small buffer layer must remain between cover foil and gel Avoid air bubbles The cover film must be positioned with the long side parallel to the VAN buffer chambers perpendicular to the arrow on the safety lid e Soak any excessive buffer from the side of the gel The gel must not swim in buffer solution Figure 17 The polyacrylamide gel has to be covered by a pre cut hydrophobic cover film A small buffer layer remains between gel and cover film Polybond film wicks slot of polyacrylamide gel TGGE System 38 e Bring the electrode wicks to an overlap with the cover film The overlap between wick and cover film should be almost 2 cm see figure 17 Avoid air bubbles e Be sure that the 2 silicone barriers are fixed to the cover glass plate before use e Cover the sandwich with the Acryl Glide treated cover glass plate see figure 18 The silicone barriers have to be positioned perpendicular to the wicks and never on top of
14. All graphics results are directly sent to the WWW client for GIF inline images and pbm images links are provided to retrieve a copy for the external viewer or for download to disk TGGE System 53 7 2 3 How to use the old Poland program Standard Poland service request form This form is an experimental service of the Biophysics Department further informations are available here The Poland server will calculate the thermal denaturation profile of double stranded RNA or DNA based on sequence input and parameter settings in this form Calculation is based on D Poland s algorithm in the implemetation described by G Steger Calculations can be done for oligonucleotides gt 15 bases or long double strands gt 50 bases respectively A form allowing for expert parameter settings is available too Graphics output is possible in Postscript HPGL GIF and PBM format numeric results are available as well Graphics results are directly sent to your WWW client For a more or less detailed description of the various parameters you may read a help page Sequence title line Sequence plain format without spacing max 180 chars per line Mismatched positions comma separated Strand concentration default 1 0e 6 M don t give the unit Thermodynamic parameters DNA default parameters Sequence length C 9 long double strand Output options __ GIF inline images bmit Click here to suomi
15. Probability of an open base pair is plotted as a function of position in sequence and temperature GelPlot Relative mobility is plotted as a function of temperature for the three different stiffness parameters MeltPlot Relative hypochromicity and its derivative is plotted as a function of temperature at 260 nm and 282 nm RNA 280 nm References for RNA Coutts S M 1971 Biochim Biophys Acta 232 94 106 Thermodynamics and kinetics of GC base pairing in the isolated extra arm of serine specific tRNA from yeast for DNA Blake R D amp Haydock P V 1979 Biopolymers 18 3089 3109 Effect of sodium ion on the high resolution melting of lambda DNA Temp50 Plot Temperature is plotted at which the corresponding base stack has a probability of 50 to be in the open state The two horizontal lines in the plot mark the temperature range of calculation i e a curve coinciding with such a line is not valid 7 3 4 3 RELATED PROGRAMS The Poland program calculates the denaturation behavior of double stranded NA LinAll RNAfold and mFold calculate the secondary structure of single stranded R NA in addition LinAll and RNAfold allow the prediction of denaturation behavior of ssRNA TGGE System 58 7 3 4 4 Restrictions The sequence has to be shorter than 1001 nucleotides but longer than 5 nucleotides Valid nucleotides are A G C U and T Calculation of asymmetric or bulge loops is not possible 7 3 4 5 Algorithm
16. Ta u L2 L3 L4 L5 le To Figure 7 upper panel Temperature profile of gradient block measured by a micro sensor on top of the gradient plate every 1mm beginning from one edge of the block 10 mm distance and 50 mm distance correspond to first L1 and last marked thick line L2 on the gradient block respectively lower panel Schematic drawing of the block TGGE System 14 The maximum temperature difference between the two sides of the gradient block T4 and T2 during electrophoresis is 45 Kelvin That means it is possible to build up a gradient between 35 C and 80 C or between 25 C and 70 C just to give two examples Programming T1 and T2 the actual temperature of L1 to L6 can be calculated by the following formula L L1 n 1 Ag L Temperature of linen n 1 6 Le L1 AG 49 Temperature difference between two thick lines 5 T u 12 13 L4 L5 le To When leaving out the gradient function the block can be cooled down to 4 C or heated up to 80 C Although Peltier elements reach lower respectively higher temperature values the surrounding plastic material does not permit the temperature range of the TGGE System to be extended The electrophoresis buffer chambers can freely be positioned around the gradient block This makes it easy to switch between parallel or perpendicular TGGE When the electrophoresis buffer chambers stay parallel to the lines of the gradient block see figure 8 left pane
17. 0 75 Na2CO 3 Dissolve 7 5 g sodium carbonate in ddH20 Total volume 1 liter Quick method for PCR products Fixation Silver Binding Developing Solution Stopping Solution 10 EtOH 0 5 Glacial Acid 100 ml ethanol and 5 ml acetic acid are adjusted with double distilled water to 1 liter 0 2 AgNO3 2 0 g AgNO is dissolved in 1 liter of distilled water Can be reused for 5 gels Store dark 3 0 NaOH 0 5 Formaldehyde Dissolve 3 g NaOH and 1 35 ml formaldehyde stock solution 37 in water in 100 ml double distilled water This solution must be prepared fresh every time identical with Fixation solution 10 EtOH 0 5 Glacial Acid TGGE System 100 Quick method using the AMRESCO SilverPAGE staining kit Code No 211 761 Fixation Sensibilisation Silver Binding Developing Solution Stopping Solution 30 EtOH 10 Acetic Acid 300 ml ethanol and 100 ml acetic acid are adjusted with double distilled water to 1 liter 30 EtOH Prepare freshly 60 ml ethanol in 140 ml double distilled water Prepare Silver Binding Agent by reconstituting contents of one pouch in 1 of ddH20 This solution must be prepared fresh every time Immediately before staining add 0 7 ml of 37 Formaldehyde to 200 ml of reconstituted Silver Binding Agent Just prior to use prepare developing solution by reconstituting contents of one pouch of Developer and 15 mg of Developer II in 200 ml of d
18. 70 82 7 Linke B et al 1995 Identification and structural analysis of rearranged immunoglobulin heavy chain genes in lymphomas and_ leukemia Leukemia 9 840 847 8 Menke M A et al 1995 Temperature gradient gel electrophoresis for analysis of a polymerase chain reaction based diagnostic clonality assay in the early stages of cutaneous T cell lymphomas 9 Hecker R et al 1988 Analysis of RNA structure by temperature gradient gel electrophoresis viroid replication and processing Gene 72 59 74 10 Baumstark T and Riesner D 1995 Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract Nucleid Acids Research 23 4246 4254 11 Loss P Schmitz M Steger G and Riesner D 1991 Formation of a thermodynamically metastable structure containing hairpin Il is critical for the potato spindle tuber viroid EMBO Journal 10 719 728 12 Riesner D 1998 Nucleic acid structures In Antisense Technology Practical Approach Series Oxford University Press p1 24 in press TGGE System 102 20 21 22 23 24 25 Wiese U et al 1995 Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis Electrophoresis 16 1851 1860 Nubel U et al 1996 Sequence heterogenities of genes encoding 16S rRNAs in Paenibaci
19. 80 139 TOGE Testkit 22 22 22 me area 93 13 1 IntroQueli nz naked 93 13 2 iPrOtOCOl ser Er a eee ered 93 13 2 1 Gel composilons ersehen cn ran 93 13 22 Runnng Duffer 222 222222222 le bel bel bla bel le bral be 94 13 2 3 Electrophoresis Paramelears nn see Res 95 13 3 KOU MAG SS eso esc rer 95 14 Appendix is ai see a at eee tease erate e e des a T a eaaa aaa enara eaan aeia 96 14 1 Technical Data eir r a a E late 96 TAT IN System aaeoa e e e elaine 96 14 1 2 Electrophoresis Chamber cccccccccseeeeeeeeeeeeeeeeeeeeesseeeeeeeeeeeeeeaeeseees 96 14 1 3 TGGE System Controller with integrated power pack 96 14 2 B lfers sense EEE RB 97 14 3 SilyerStaining Solutions Sa ee ek re rkeee lenken kgerkdee tere 99 13 References Eee 101 16 Order information and spare Parts ccceccceeeeeeeeeeeeeeeeeeeeeeeeeeeeneeeeeeeeeees 105 17 Instructions for return shipment uu 444444044000nnnnnnnnnnnnn nenn nun nnnnnnnnnne 106 TGGE System 18 Equipment Decontamination Certificate uuuun0000nnnnnnnnnnnnnnnnnnnnnnnnnn 19 Warranty 2222222222 ea ea Declaration of Conformity TGGE System 4 1 The TGGE System 1 1 Introduction Temperature Gradient Gel Electrophoresis is a new and powerful electrophoresis method for separation of nucleic acids like DNA or RNA or for analysis of proteins The TGGE method which is covered by patents uses the temperature
20. Electrodes are dirty or 4 Check clean the electrodes inside damaged the buffer chambers Wicks to dry and or 5 Immerse the electrophoresis placed not correct wicks in the buffer and place them properly on the gel Programmed voltage to 6 Increase voltage low Extremely high current High ionic strength in 1 Check the composition of gt 50mA electrophoresis buffer electrophoresis buffer and gel wrong buffer concentration Silver staining artifacts Cloudy yellow or brown 1 Wash extensively after silver staining binding step 2 Use rocking table for staining protocols 3 Place gel up side in the staining tray use sufficient solution Brown spots in the gel Un dissolved crystals of 1 Dissolve urea completely before urea remain in the gel gel pouring Contaminated chemicals 2 Use fresh stock solutions Filter prior to use Wear only non powdered gloves during handling the gel Gel casting glass plate 3 Clean gel casting plate and the or Cover film have not been cleaned properly Cover film carefully before they come in direct contact with the gel Wear gloves when handling the gel TGGE System 83 Problem Cause Solutions Gel is stained completely black or looks like a silver mirror _ Chloride ions are contaminating the staining solutions the electrophoresis buffer or the gel solution During the staining protocol silver chloride AgCI is precipitated
21. J A Sugimoto N Caruthers M H Neilson T amp Turner D H 1986 Proc Natl Acad Sci USA 83 9373 9377 Improved free energy parameters for predictions of RNA duplex stability e for RNA in 1M NaCl P rschke D Uhlenbeck O C amp Martin F H 1973 Biopolymers 12 1313 1335 Thermodynamics and kinetics of the helix coil transition of oligomers containing GC base pairs e DNA e for DNA in 0 019 M NaCl Gotoh O 1983 Adv Biophys 16 1 52 Prediction of melting profiles and local helix stability for sequenced DNA efor DNA e in1MNaCl Breslauer K J Frank R Bloecker H amp Marky L A 1986 Proc Natl Acad Sci USA 83 3746 3750 Predicting DNA duplex stability from the base sequence e e for DNA in 0 1 M NaCl Klump H H 1987 Canad J Chem 66 804 809 Energetics of order order transitions in nucleic acids TGGE System 62 Klump H 1990 in Landolt B rnstein New Series Group VII Biophysics Vol 1 Nucleic Acids Subvol c Spectroscopic and Kinetic Data Physical Data I W Saenger ed Springer Verlag Berlin p 244 245 Calorimetric studies on DNAs and RNAs e for DNA in 1M NaCl SantaLucia J Jr Allawi H T amp Seneviratne P A 1996 Biochemistry 35 3555 3562 Improved nearest neighbor parameters for predicting DNA duplex stability for DNA in 1 M NaCl Allawi H T amp SantaLucia J Jr 1997 Biochemistry 36 10581 10594 Thermodynamics and NMR of Internal G T Mismatch
22. Pretreatment of glass plate with spacer and slot former Glass plate with spacer and slot former must be carefully treated with Acryl Glide solution Order Number 211 319 or a similar hydrophobic solution Drop about 0 5 ml of solution onto the plate and especially between the slot former This protection layer helps to withdraw the polyacrylamide gel from the sandwich after polymerization Wait 2 3 min and than polish the plate with soft paper to remove any haze This procedure should be repeated after each run Do not drop Acryl Glide onto the spacer of the glass plate This possibly leads to leakage during polymerization Clean spacers with ethanol before assembling the glass plate sandwich If necessary treat the spacers with a small amounts of silicone grease to protect leakage Polybond film Use only original pre cut Polybond film which perfectly fits onto the gradient block Biometra recommends to use Polybond film only once Repeated usage and especially staining might weaken the strength of the Polybond film The Polybond film has two different sides one hydrophobic side which repels water and one hydrophilic side on which a water drop will adhere You can test the different sides with a drop of water The protection paper is attached to the hydrophobic site Remove the protecting paper sheet before assembling the sandwich Handle the Polybond film only with powder free gloves The hydrophobic side of the Polybond film
23. Run the electrophoresis at 200 undergone diffusion 300 V depending on the buffer inside the gel because used of extremely prolonged electrophoresis time at low voltage 6 The DNA has Incubate the gel directly after the undergone diffusion inside the gel because the DNA has not been fixed after running the gel Only in parallel TGGE 7 The band has migrated to a temperature which causes an irreversible transition of the DNA into single strands run in Fixation solution of the silver staining protocol 10 EtOH 0 5 acetic acid Silver stain Check the melting behavior of your DNA fragment in a perpendicular TGGE gel If perpendicular TGGE shows a sigmoidal S shaped curve determine the effective range of separation Set up new conditions for parallel TGGE If parallel TGGE does not show a sigmoidal S shaped curve check all items listed under No S shaped curve in perpendicular TGGE TGGE System 85 Problem Cause Solutions Band pattern is disturbed or distorted PAS Air bubbles in the gel Run a new gel without air bubbles 2 Gelhas been punctured 2 Load the sample carefully Don t pipette tip during touch the gel by the pipette tip loading of the sample 3 The edges of the gel 3 Carefully protect the gel against have dried out during evaporation Cover the gel electrophoresis The additionally with the special Cover front with t
24. attention to the position of electrode wicks on top of the polyacrylamide gel Polybond film pre cut Polybond film slot of polyacrylamide gel e Avoid any contact between sample slots and electrode wicks Otherwise the samples will diffuse into the electrode wicks e Load the samples quickly at room temperature without air bubbles Do not start the temperature gradient the temperature gradient is established after the samples have fully entered the polyacrylamide gel TGGE System 41 Depending on the slot size of the used gel Biometra recommends the following amounts of material glass plate with 8 slots glass plate with 12 slots 5 ul volume 3 5 ng DNA RNA of interest 3 ul volume 1 3 ng DNA RNA of interest glass plate with 18 slots 2 ul volume approx 1 ng DNA RNA of interest If less volumes fill up the slots with running buffer or loading buffer to the volumes listed on top This will create better results The time between mounting the gel onto the gradient block and loading the sample must not exceed 5 minutes Close the safety lid of the electrophoresis chamber and start electrophoresis at 20 C or 25 C and 250 V for 2 5 min Standard electrophoresis conditions are given in chapters 5 3 and 6 1 Make sure that the orientation of the gel and the safety lid is exact as indicated in the following oO al fT Wait at the electrophoresis chamber un
25. et al Use only with stacking parameters according to Turner et al p t TGGE System 63 Therefore Sigma influences the cooperativity and the half width of each transition With a f you can change the default algorithm only in case I p With the original algorithm of Poland default computing time is proportional to the square of the sequence length With a f the modified algorithm of Fixman amp Freire is used which results in computing time proportional to 10 times the sequence length but works only with loop parameters according to Poland I p up to a sequence length of 1000 base pairs References Poland D 1974 Biopolymers 13 1859 1871 Recursion Relation Generation of Probability Profiles for Specific Sequence Macromolecules with Long Range Correlations Fixman and Freire 1977 Biopolymers 16 2693 2704 Theory of DNA melting curves Gralla J amp Crothers D M 1973 J Mol Biol 78 301 319 Free energy of imperfect nucleic acid helices III Small internal loops resulting from mismatches Freier S M Kierzek R Jaeger J A Sugimoto N Caruthers M H Neilson T amp Turner D H 1986 Proc Natl Acad Sci USA 83 9373 9377 Improved free energy parameters for predictions of RNA duplex stability e TEMPERATURE RANGE OF CALCULATION The temperature range for calculations has to be adapted to the other parameters thus see the topic Suggestions In principal not more than 110 temperature
26. in the gel gel looks milky and is then reduced to elemental silver a Check if tap water has been used for one of the buffers tap water always contains chloride ions Prepare and use only fresh solutions with ddH20 b Check if tap water has been used for washing the gel twice after incubation with 0 1 AgNO in dest H20 Use ddH O instead c If deionized water is used its integrity should be checked no chlorid ions Take approximately 1 ml ofthe deionized water or the buffer you want to check and add some drops of 0 1 AgNO3 If you see a milky precipitation silver chloride AgCl the solution is contaminated with chloride ions Use distilled water for preparing the buffers and washing 2 High amounts of gel chloride ions Desalt the sample prior to TGGE contaminate the sample Gel has a heavy background 1 Heavy background Check the purity ofthe sample caused by smearing of priorto TGGE the sample High amounts of proteins or polysaccharides also stained by the silver may contaminate the sample 2 Old chemicals Use fresh stock solutions especially acrylamide bis solution 3 Silver staining protocol Follow the silver staining protocol has been carried out as exactly as described Use an incorrectly excess of freshly distilled water when washing the gel twice Do not prolong the incubation in Developing solution No DNA bands are visible in 1 No DNA or amount of Check the amounts of DNA the gel D
27. one end to the other but by cooperative denaturation of long stretches called melting domains The length of a melting domain is 25 to several hundred base pairs The midpoint melting temperature Tm and the length of a melting domain are mainly determined by the nucleotide sequence of the DNA The Tm of DNA fragments differing by even small changes such as point mutations can differ by as much as 1 5 C When heteroduplices hybrids of two species of DNA fragments differing in their base composition have been formed the mismatches lower the Tm value significantly Thus the heteroduplex analysis is the preferable because of the additional resolution provides 1 28 The principle by which TGGE uses differences in Tm is that the DNA fragments are electrophoresed through a linear temperature gradient in the polyacrylamide gel When the fragments reach the temperature at which the lowest melting domain starts to melt they take on a branched Y shaped configuration which slows down mobility in the TGGE gel matrix The electrophoretic migration of fragments differing by single base changes is retarded by branching at different temperatures thus they are resolved from one another during temperature gradient electrophoresis The denaturing behavior of any DNA fragment can be predicted if its sequence is known For this purpose the POLAND calculation can be used The POLAND software is available in the internet http www biophys uni duesseldorf de ser
28. points are allowed for a single calculation e MISMATCHED POSITIONS in original sequence Mismatches are given as a comma separated list of sequence positions f e m 2 3 111 specifies mismatched base pairs at positions 2 3 and 111 If the mismatch is longer than a base pair the position of each base has to be given separately The sequence position of the mismatched base pair s may be given in any order Calculation of asymmetric or bulge loops is not possible these have to be modeled by larger mismatches internal loops e CONCENTRATION and DISSOCIATION CONSTANT c 1 e 6 Concentration of single strands CO n 1 e 3 Dissociation constant R amp cO influences temperature Tm and half width of the second order transition i e the strand separation The dissociation constant B has to be in the range 1 gt R gt 1 E 5 and the strand concentration has to be in the range 1 gt CO gt 1 E 13 If case of short oligonucleotides R might be calculated according to Benight A S amp Wartell R M 1983 Biopolymers 22 1409 1425 Influence of base pair changes and cooperativity parameters on the melting curves of short DNAs and Benight A S Wartell R M amp Howell D K 1981 Nature 289 203 205 Theory agrees with experimental thermal denaturation of short DNA restriction fragments TGGE System 64 e STIFFNESS of nucleic_acid Lr Stiffness of nucleic acid or gel pore size is given f e with 1 40 90 200
29. stop C quit D To stop a current running program you press B Elpho and again D stop By pressing SC quit you return to previous display without any changes When pressing amp D stop the run will be aborted and you leave the actual running program Pressing B pause holds the actual situation of the gradient stopping electrophoresis holding the temperature gradient pause is blinking and shown in the display alternatively with the time Pressing SB Elpho in pause status Pressing B contin will continue the program TGGE System 22 Function key A T1 22 0 C T2 22 0 C block off A B Elpho C programs D B start stop pause C edit delete copy D special functions A B Elpho C programs D L1 25 0 C L6 60 0 C hold 1 Om 1s 1 16Vh El 250 V 8mA 20W A list B block C program D T1 21 1 C T2 63 8 C L133 21 1 32 0 39 0 L4 6 46 0 53 0 60 0 lt const rtime h m Pressing A in the main menu results in the following display Pressing A in a running program results in the following dis play The actual temperatures of T1 and T2 as well as the actual tem peratures of L1 to L6 are shown in the display The limiting factor const V mA or W is indicated by an blinking arrow lt The actual remaining electrophoresis time is shown on the bottom right side of the display TGGE System 23
30. the adequate fragment length of the amplicon This program gives a rough estimation too what temperature parameters will fit best to the desired separation gt A revised version of the POLAND program can be found on the world wide web http www biophys uni duesseldorf de service polandform htm For example the polymorphic site is represented by allele A and allele a The two alleles can either exist as homoduplices AA or aa or heteroduplices Aa or aA see figure 3 Sequence of Sequence of Allele A Allele a a DOOD gt D P QOQQ A gt DO gt oQO gt AA Aa aa a A Figure 3 Schematic drawing of double stranded DNA with polymorphism A or a left panel and corresponding DNA sequence right panel Each line represents double stranded DNA The TGGE System used as perpendicular TGGE see above figure 2 gives the possibility to identify the different alleles by their individual melting behavior Samples with homoduplex AA or aa have a distinct melting temperature e g Tm and Tmo see figure 4 at which double stranded DNA separates into branched DNA At even higher temperature the branched DNA separates into individual strands After perpendicular TGGE a heteroallele sample like Aa normally shows four different Tm values The PCR amplification of a heteroallele sample results in four different double stranded DNA types The two homoduplices AA and aa as well as two hetero
31. the gradient block Sandwich of polyacrylamide gel pre cut cover foil electrode wicks and cover glass plate with silicone barriers during perpendicular TGGE run Polybond film slot of polyacrylamide gel TGGE System 43 Start the temperature gradient and wait until the gradient has been established usually 0 5 1 minute Start the electrophoresis run The Bromophenol blue dye only gives you an indication how far the samples have migrated until you have optimized the best run time After the electrophoresis run switch off the controller open the safety lid of the electrophoresis unit remove the polyacrylamide gel and proceed further for staining the gel chapter 6 5 It is recommended to fix the gel immediately in order to improve the analysis TGGE System 44 6 5 Silver staining Aside from autoradiography silver staining is the most sensitive method for detecting small amounts of DNA RNA or proteins in polyacrylamide gels Due to the low thickness of the gels 0 5 mm the staining procedure takes no more than 35 minutes Other staining protocols may be used but generally exhibit less sensitivity This must be considered in relation to the amount of DNA loaded on the gel All incubation steps are done in small plastic containers which are agitated on a rocking platform e g order number 042 400 or 042 500 For handling several polyacrylamide gels simultaneously Biometra offers a semi auto
32. the more precise Volt x hour integration Pressing B Time replaces V H by time El Three different electrophoresis parameters voltage current or wattage can be set Current and wattage are pre set at max values of 500 mA and 30W respectively In the beginning we recommend to set only the Voltage Depending on the resistance of the gel electrophoresis the controller will regulate the other two parameters automatically After confirming the voltage by pressing SD enter and pressing two times SD gt the following two parameters are not changed Ramptime Ramping time Pressing B special gives you the choice to choose how fast the Gradient block is going to the established gradient Normally you choose 1s which means maxi ramping speed In this case enter 1 and confirm with amp D enter Pressing B standard results in the standard display Pressing D gt starts the programming of step 2 in this program By pressing SC quit any change will be saved and the following messages appear After a few seconds the main menu appears TGGE System 21 Function key B Start Stop Start block function and electrophoresis Start program __ Alist Bdel C quit D enter L1 25 0 C L6 60 0 C ramp 1 time Om 1s El 250V 20mA 20W A list B block C program D L1 25 0 C L6 60 0 C hold 1 Om 1s 0 00 Vh El 250 V 8mA 20W A list B block C program D
33. the wicks Figure 18 Side view of the polyacrylamide gel 8 on top of the gradient block Sandwich of polyacrylamide gel pre cut cover foil electrode wicks and cover glass plate with silicone barriers during perpendicular TGGE run Polybond film slot of polyacrylamide gel e Start the temperature gradient and wait until the gradient has been established usually 0 5 1 minute e Start the electrophoresis run e The Bromophenol blue dye only gives you an indication how far the samples have migrated until you have optimized the best run time e After the electrophoresis run switch off the controller open the safety lid of the electrophoresis unit remove the polyacrylamide gel and proceed further for staining the gel chapter 6 5 It is recommended to fix the gel immediately in order to improve the analysis TGGE System 39 6 4 Parallel TGGE During parallel TGGE a mixture of molecules is separated over a narrow temperature range determined by perpendicular TGGE The temperature gradient is parallel to the electrophoresis run direction Steps before TGGE sample preparation refer to chapter 4 1 programming of temperature gradient and electrophoresis parameters refer to chapter 3 2 2 To prepare in advance polyacrylamide gel attached to Polybond film running buffer 250 ml for each chamber pre cut and pre soaked electrode wicks pre cut cover foil glass plate treated on both sides with Acr
34. 854 70 0 IS X S 904 62 0 AN 2 54 0 NZ 371 P LO I S NG a 704 0 3 654 i 3 604 0 4 fad 0 2 gt 0 04 D T 100 50 T T DD 20 40 50 cn 7o s0 90 100 110 120 Stack index Sequence Optimized DNA fragment GC clamp attached Domains distinctly different Two distinct second order line plateaus Poland analysis of Withgc_18_28 4 9 on September 23 1998 18 28 37 77 Poland analysis of Withgce_18_28_34 q on Se Sequence vs PrfbabMity vs Temperature Stack index vs Temp of 50 probability er 23 1998 18 28 48 33 first order second order 905 854 804 754 704 654 604 Temp of 507 probabilityLC 554 50 s0 90 100 110 120 130 140 10 20 30 40 so 60 70 Stack index Sequence Optimized DNA fragment with mismatch Mismatch position Mismatch changes melting behavior Poland analysis of Miffmat ch_18_29_34 Seq on September 23 1998 18 29 38 55 Poland analysis of Mismatch_1849_34 Seq on September 23 1998 18 29 49 73 Segflence vs Probability vs Temperature Stack index vs Temp of SOgProbability first order second order y EEE u Temp of 507 probabilityLC N
35. AND program completely single stranded DNA 5 Check the gradient block 5 Unstable temperature Purge any air bubbles from the gradient or no temperature gradient at all gradient block TGGE System 86 no temperature gradient at all e wrong orientation temperature gradient parallel to the electric field e fragment is not stabilized by a GC rich part GC clamp is required instable temperature gradient only ssDNA has been loaded ineffective renaturation DNA has not been melted ionic strength in the gel is too high amount of urea is insufficient Figure 25 No S shaped curve in perpendicular TGGE TGGE System 87 Problem Cause Solutions PCR product derived from a putative heterozygous locus exhibits no heteroduplex bands in parallel TGGE fig 26 a and b Electrophoresis time is too short The temperature gradient range used in the experiment is not sufficient The temperature at the cathode is too high The melting domain containing the point mutation has already been denatured when the DNA enters the gel Masking of homoduplex bands Only heteroduplex bands are visible in the gel The DNA fragments have already passed the effective range of separation Homo and heteroduplices have been separated but the fastetst running homoduplex bands have also passed Tdiss the temperature of total denaturation Due to the irrev
36. Acid Stock solution 10 x conc 20 mM EDTA 890 mM TRIS Do not titrate to adjust pH TBE Running buffer 0 1 x conc TBE up to 1x conc TBE is possible Na TAE pH 8 4 1 M Sodium acetate Stock solution 10 x conc 10 mM EDTA 400 mM TRIS pH 8 4 titrate with Acetic Acid never use HCI Na TAE Running Buffer 0 2x conc Na TAE pH 8 4 ME MOPS EDTA 1M MOPS Stock solution 50 x conc 50 mM EDTA pH 8 0 ME Running Buffer 1 x conc ME pH 8 0 SSCP buffer SSCP ME buffer 1M MOPS Stock solution 50 x conc 250 mM EDTA Free Acid pH 8 0 SSCP ME Running buffer 1 x conc SSCP ME pH 8 0 Others TE buffer 10 mM Tris HCl 0 1 mM EDTA pH 8 0 TEMED Solution of N N N N tetramethylethylendiamine APS 4 Ammonium persulfate Glycerol 40 40 glycerol in water Glycerol 50 50 glycerol in water TGGE System 99 14 3 Silver staining solutions Standard method Fixation Silver Binding Developing Solution Stopping Solution 10 EtOH 0 5 Acetic Acid 100 ml ethanol and 5 ml acetic acid are adjusted with distilled water to 1 liter Prepare freshly 0 19 AgNO3 1 9g AgNO is dissolved in 1 liter of distilled water Can be reused for 5 gels Store dark 1 5 NaOH 0 08 NaBH 0 1 Formaldehyde Dissolve 15 g NaOH in 1 liter distilled water and add 0 8g NaBH Immediately before developing add 2 7 ml formaldehyde stock solution 37 in water This solution must be prepared fresh every time
37. Biometra a Whatman company TGGE System 230 V Code No 024 000 115V Code No 024 090 Manual March 1999 Warning J Please read these instructions carefully VAN before using this apparatus Biometra biomedizinische Analytik GmbH Rudolf Wissell Stra e 30 D 37079 G ttingen P O Box 1544 D 37005 G ttingen Tel 49 0 5 51 50 68 6 0 Fax 49 0 5 51 50 68 6 66 e mail info biometra co uk internet http www biometra de TGGE System The TGGE method is covered by patents issued to Diagen now QIAGEN GmbH The polymerase chain reaction PCR process is covered by patents issued to Hoffmann La Roche Acryl Glide is a trademark of Amresco Inc Biometra is a trademark of Biometra GmbH Whatman is a trademark of Whatman International Ltd The POLAND software service established by Gerhard Steger Department of Biophysics University of Duesseldorf is available by internet http www biophys uni duesseldorf de service polandform html Manual Version 2 5 Software Version 2 10 2 11 7 24 2002 Biometra GmbH Rudolf Wissell Str 30 37079 Gottingen Germany TGGE System 1 Table of Contents 1 THE TGGE System 2 2 22 2 2 2 4 121 Introduction seele ses else ont orients 4 1 2 Prineipleol MEIN Giaceatatortetatctstettelsclatetatet tet ee 4 1 3 Special features of the TGGE System uuunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn 5 1 4 How to start with TGGE analysis
38. GE System 115 V dito 024 090 TGGE System Controller with integrated power supply 230 V 024 001 TGGE System Controller with integrated power supply 115 V 024 091 TGGE electrophoresis unit with Peltier element powered gradient block 2 removable 024 002 electrophoresis buffer chambers and connector cable TGGE connector cable controller to electrophoresis unit 024 033 TGGE Starter Kit with 3 Bonding glass plates 3 types of glass plates with slots pre 024 003 cut electrode wicks pre cut Polybond film and cover film TGGE Testkit samples for 4 parallel and 4 perpendicular TGGE runs 20 ul wild 024 050 type 20 ul mutant 120 ul Heteroduplex 180 ul loading buffer with blue marker for Na TAE buffer Accessories TGGE removable electrophoresis buffer chambers 2 pcs 024 010 TGGE electrode wicks pre cut 8 x 7 cm 100 pcs 024 015 TGGE bonding plate 9 x 9 cm w o spacer 024 021 TGGE glas plate 9 x 9 cm 8 slots 4x3x0 4 mm approx 5 ul 024 022 TGGE glas plate 9 x 9 cm 1 slot rectangular 40x3x0 4 mm approx 50 ul 024 023 TGGE glas plate 9 x 9 cm 1 slot diagonal 62x3x0 4 mm 024 024 approx 75 ul TGGE glas plate 9 x 9 cm 12 slots 3x2x0 4 mm approx 3 ul 024 025 TGGE glas plate 9 x 9 cm 18 slots 2x2x0 4 mm approx 2 ul 024 026 TGGE glas plate 9x 9 cm 0 5 mm spacer no slots 024 027 TGGE Polybond film pre cut 8 8 x 8 8 cm 25 pkg 024 030 TGGE Cover glass plate with silicone barriers 024 031 10 cover films pre cut 7 x 6 cm TGGE cover f
39. NA sample is below the level of detection 2 Too much DNA inverse If bands contain high amounts of silver staining DNA the silver staining may result in an inverse staining the background is darker than the band itself Reduce amounts of DNA Gel fades out Stopping solution 0 75 NazCO3 was not sufficient Incubate the gel in the staining buffer for 10 min TGGE System 84 Problem Cause Solutions Band pattern in general Bands are diffuse Sample volume is too large Reduce amount of DNA RNA 2 Sample is contaminated Purify the sample in order to reduce contaminating proteins or polysaccharides 3 The DNA has Proceed with silver staining undergone diffusion protocol immediately after inside the gel because electrophoresis the DNA has not been fixed after running the gel Interpretation of the TGGE band pattern DNA bands are diffuse 1 Sample volume is too Load the correct sample volumes large 2 Too much DNA in the Check the amount of DNA sample gel is overloaded 3 Temperature gradient Check the thermal coupling has not been stable solution used Use 0 1 Triton or during electrophoresis Tween 20 4 The gel has been shifted Check the volume of thermal during electrophoresis coupling solution used The gel thus the temperature should not change the position gradient inside the gel during the run has not been stable 5 The DNA has
40. Please enclose a precise description of the fault which also reveals during which procedures the fault occurred if possible e Important Clean all parts of the instrument from residues and of biologically dangerous chemical and radioactive contaminants Please include a written confirmation use the Equipment Decontamination A N Declaration following on the next page that the device is free of biologically dangerous and chemical or radioactive contaminants in each shipment If the device is contaminated it is possible that Biometra will be forced to refuse to accept the device e The sender of the repair order will be held liable for possible losses resulting from insufficient decontamination of the device e Please enclose a note which contains the following a Sender s name and address b Name of a contact person for further inquiries with telephone number TGGE System 107 18 Equipment Decontamination Certificate To enable us to comply with german law i e 828 StrlSchV 17 GefStoffV and 819 ChemG and to avoid exposure to hazardous materials during handling or repair will you please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS TEL NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Has the equipment been cleaned and deco
41. System 78 11 TGGE in protein analysis TGGE can be successful applied to investigation of protein Structural transitions and also thermostability of protein nucleic acid interactions In comparison to conventional methods such as spectroscopy hydrodynamics or calometry TGGE analysis offers several advantages e Only minimal amounts of sample material are required e TGGE may be carried out by using crude protein extracts e The effect of additives that influence the protein stability may easily be investigated 11 1 Buffers In contrast to nucleic acids which can generally all be analyzed with standard buffer conditions each protein requires its own special buffer system This buffer has to fulfill the same requirements as those for native gel electrophoresis of proteins e The protein has to be in its native conformation at low temperature e g room temperature e The protein has to carry a net charge e The protein has to be soluble in the used buffer e The protein has to migrate as a honogeneous band under native conditions This single one prerequisite has to be tested on a non denaturing gel before testing the denaturation behavior on TGGE Additionally e The pH value of buffer should be virtually independent of the temperature e In order to avoid excessive current in TGGE gel above 100 mA the ionic strength of the electrophoresis buffer should not exceed 30 mM TGGE System 79 Temperature dependence o
42. and 5 ml acetic acid are adjusted with double distilled water to 1 liter Prepare freshly 0 2 AgNO 2 0 g AgNO is dissolved in 1 liter of distilled water Can be reused 5 times Store dark 3 0 NaOH 0 5 Formaldehyde Dissolve 3 g NaOH and 1 35 ml formaldehyde stock solution 37 in water in 100 ml double distilled water This buffer must be freshly prepared immediately before use identical with Fixation solution 10 EtOH 0 5 Glacial Acid TGGE System 47 Quick method using the AMRESCO SilverPAGE staining kit Code No 211 761 Step Time Solutions Notes Fixation 15 min 2x 100 ml Fixation solution Sensibilisation 10 min 2 x 100 ml 30 ethanol prepare freshly Washing 10 min 3 x 200 ml fresh ddH20 Demineralised water may be ok Silver Binding 15 min reconstituted Silver Binding prepare freshly Agent Formaldehyde Washing 0 5 1 Rinse under fresh ddH20 min Developing 1 2 min reconstituted Developing prepare freshly solution Formaldehyde Develop to desired level Stopping 5 min 7 5 Acetic acid Preparing for 1 5 h 50 glycerol Not absolutely storage necessary Drying Room temperature Preparing the gel for storage 1 5 h at room temperature in 50 glycerol is not necessary Staining solutions Fixation Sensibilisation Silver Binding Developing Solution Stopping Solution 30 10 EtOH Acetic Acid 300 ml ethan
43. aphics do you want Br m m m m Graphics size 7x7 doi GIF format z Click here to or Institut f r Physikalische Biologie Reset Click here to the form to defaults Department of Biophysics Heinrich Heine Universit t D sseldorf Germany Feb 26 1999 G Steger M Labensky A J ger TGGE System 66 7 4 The optimized DNA fragment The optimized fragment for detection of point mutations in dsDNA is derived by PCR amplification and has a length of 200 300 to max 800 900 bp It consists of 1 2 melting domains derived from the native sequence plus a synthetic stabilizing region GC clamp The GC clamp is highly stable because of 3 hydrogen bridges between G and C whereas there are only 2 hydrogen bridges between A and T This clamp may either consist of a 40 bp artificial stretch of GC base pairs 29 or a covalent chemical clamp Psoralen Furo 3 2 g coumarin C11H603 Psoralen is the better choice for temperature gradients at high temperatures because Psoralen intercalates with the double helix and after UV treatment it links both strands covalently irreversible binding Both clamps are introduced into the DNA fragment by a 5 overhang of one of the PCR amplification primers For easier reading of the following text both kinds of clamps will re referred to as GC clamps further on The melting properties of a DNA fragment are best described by the two dimensional TempPlot
44. as normally used for parallel TGGE is the fastest approach to determine the optimal conditions for SSCP Proceed as described for parallel TGGE Establish the temperature gradient from 5 C cathode black to 30 C anode red and use SSCP ME buffer for the run Run at 200 V for 2 5 hours Covering the gel with a protective cellophane sheet is not absolutely necessary because the gel will not dessicate at the temperatures used in this protocol Silver stain the gel 9 5 Routine analysis When the temperature range of SSCP separation has been determined routine analysis can be performed in a gel with constant temperature TGGE System 76 10 TGGE in RNA analysis TGGE is a perfect tool to analyze RNA for secondary structures Applications have been published on the differentiation of plant pathogen variants 1 33 34 35 36 37 analysis of intermediates of plant pathogens 1 11 30 31 32 33 and also on the analysis of hairpin structures in m RNA 1 38 The technique of choice is perpendicular TGGE Depending on the species of RNA M RNA r RNA etc which is to be analyzed standard protocols and conditions described in this manual are generally applicable but may require minor modifications 10 1 Completely double stranded RNA The Tm values of a dsRNA sequence will be about 20 C higher in comparison to the corresponding ds DNA sequence For dsRNA with low GC content start with the standard protocols using ME buffer Fo
45. cccc ccgcg cccgg cgccc ccgc 7 4 2 Chemical clamp with Psoralen Furo 3 2 g coumarin C11H603 The 5 end of the primer for the end of the segment at which a clamp is optimal POLAND program must carry a appropriately linked psoralen moity at the end adjacent to T or A preceding the genomic sequence The optimal primer sequence may be 5 Pso pTaPpnpnp 3 given the preference of psoralen for binding between TpA and ApT pairs 7 Crosslinking of the PCR product is done e g ina flat bottom microtiter plate using a 365 nm UV source Working with small volumes it may be necessary to minimize evaporation by cross linking at 4 10 C The yield is not affected by temperature The distance of the sample from the UV source affects the yield 15 min at 0 5 cm distance of the sample from an 8 W UV lamp is sufficient TGGE System 69 7 4 3 POLAND analysis of samples Unoptimized DNA fragment Domains not distinctly different Second order line is flat Poland analysis of Withoitge Vi 30_43 Seq on September 23 1998 18 30 47 54 Poland analysis of Withoutgc_18_30_43 Seq on Septemer 23 1998 18 30 57 16 Wrobability vs Temperature Stack index vs Temp of 50 probability first order second order TECI 907 f
46. ch build up the temperature gradient during electrophoresis can be found Both sides of the gradient block can reach any preset temperature from 15 C 80 C The orientation of the temperature gradient i e which side of the gradient shall be cold or hot can be freely determined Between symbols T4 and Tz six thick lines L1 to L6 and five thin lines not coded are marked on the block which represent the entire linear range of the gradient block see figure 7 The temperature difference between two lines is identical from line to line E g if T4 is 30 C and T2 is 75 C the temperature difference is 7 4 C between two thick lines or 3 7 C between a thick and a thin line When performing parallel TGGE the beginning and the end of the linear temperature gradient are represented by the first and the last line Whereas when performing perpendicular TGGE the ends of each line represent the linear temperature range When choosing a temperature gradient e g between 25 C and 65 C these two temperatures can actually be found at the first marked line figure 7 10mm distance to the block edge and at the last marked line figure 7 50 mm distance on the block The block areas to the left and right of these lines are slightly hotter respectively cooler see figure 7 and table 1 2 TGGE System 13 Linearity of block 60 a oO 40 20 10 Temperature C 1 6 11 16 21 26 31 36 41 46 51 56 61 Distance mm
47. cm perpendicular 22 5 cm x 22 5 cm x 23 cm 4 2 kg 14 1 3 TGGE System Controller with integrated power pack Program stores Display Languages Mains voltage Frequency Wattage Fuses Interfaces Size LxWxH Weight Integrated power pack Voltage Current Power 100 LCD German English 115V 230 V 50 60 Hz max 30 VA 2x 3 15 AT 115 V 2x 1 6 AT 230 V 1 parallel port Centronics for printer 1 serial port RS232 31 cm x 22 cm x 11 5 cm 3 8 kg max 400 V max 500 mA max 30 W TGGE System 97 14 2 Buffers Loading buffers Loading buffer TBE 0 1x conc TBE up to 1x conc TBE is possible 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye Loading buffer Na TAE 0 2x conc Na TAE pH 8 4 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye Loading buffer ME 10 x conc MOPS 10 mM EDTA 0 05 Bromophenol Blue dye 0 05 Yxlene Cyanol dye pH 8 0 Denaturation Renaturation DR Loading buffers DR Loading buffer TBE 0 1x conc TBE up to 1x conc TBE is possible 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye 7M Urea DR Loading buffer Na TAE 0 2x conc Na TAE pH 8 4 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye 8M Urea DR Loading buffer ME 20x conc MOPS 20 mM EDTA 0 01 Bromophenol Blue dye 0 01 Yxlene Cyanol dye 8M Urea pH 8 0 TGGE System 98 Running buffers TBE 890 mM Boric
48. d high degree of mismatches This concentration increases Tm instead of the standard concentration 8 M urea by approx 16 20 C using TBE buffer or 8 12 C using Na TAE buffer 10 M urea can be used for high GC and lowers Tm instead of the standard concentration 8 M urea by approx 8 10 C using TBE buffer or 4 6 C using Na TAE buffer Glycerol reduces the steepness of very cooperative transition curves broadening the profile and expanding the temperature range for detecting small Tm differences of closely related nucleic acids 0 Glycerol increases cooperativity gt 200 bp narrower transitions gt 2 Glycerol lowers cooperativity gt 200 bp broader transitions TGGE conditions Voltage can be raised to 400 V if the current is below 30 mA Current should not exeed 30 mA Running buffer pre run run 0 2 x conc Na TAE 250 V 10 12 mA 2 5 min 250V 15 20 mA 30 60 min 0 1 x conc TBE 250 V 4 5mA 2 5min 250V 9 12 mA 30 60 min T1 20 C can be raised to obtain optimized resolution T1 20 C lowering should be avoided by using 4M urea T2 60 C higher temperatures should be avoided by using 10 M urea or and 10 mM MOPS T2 60 C can be lowered to obtain optimized resolution T2 80 C maximum temperature can be used if the gel is carefully protected against evaporation Sample preparation Denaturation renaturation cycle e Renaturing at 50 C higher temperatures higher stringency can be chose
49. d replication and processing Gene 72 59 74 Jiang L Chen W Tain L P and Liu Y 1991 Temperature gradient gel electrophoresis of apple scar skin viroid Acta Microbiol Sin 30 278 283 Riesner D Hecker R and Steger G 1988 Structure of viroid replication intermediates as studied by thermodynamics and temperature gradient gel electrophoresis In Sarma R H and Sarma M H eds Structure amp Expression Vol I From Proteins to Ribosomes Adenine press 261 285 Riesner D Steger G Zimmat R Owens R A Wagenhofer M Hillen W Vollbach S and Henco K 1989 Temperature gradient gel electrophoresis of nuleic acids Analysis of confor mational transitions sequence variations and protein nucleic acid interactions Electrophoresis 10 377 389 Rosenbaum V and Riesner D 1987 Temperature gradient gel electrophoresis thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extract Biohys Chem 26 235 246 Sch nborn J Oberstra J Breyel E Tittgen J Schumacher J Lukacs N 1991 Monoclonall antibodies to double stranded RNA as probes of RNA structure in crude nucleic acid extracts Nucleic Acids Res 19 2993 3000 Po Tien Steger G Rosenbaum V Kaper J and Riesner D 1987 Double stranded cucumovirus associated RNAS experimental analysis of nec rogenic and non necrogenic variants by temperature gradient gel electrophoresis Nucleic Acids Res 15 5069 5083 TGGE Syste
50. dH20 This solution must be prepared fresh every time Immediately before developing add 0 7 ml of 37 Formaldehyde to 200 ml of reconstituted developing solution 7 5 Acetic Acid 75 ml acetic acid are adjusted with double distilled water to 11 TGGE System 101 15 References 1 Riesner D Henco K and Steger G 1990 Temperature Gradient Gel Electrophoresis A method for the analysis of conformational transitions and mutations in nucleic acids and protein Page 169 250 In Chrambach A Dunn M J Radola B J Advances in Electrophoresis Vol 4 VCH Verlagsgesellschaft Weinheim 2 Kappes S et al 1995 p53 mutations in ovarian tumors detected by temperature gradient gel electrophoresis direct sequencing and immunohistochemistry Int J Cancer 64 52 59 3 Milde Langosch K et al 1995 Presence and persistence of HPV and p53 mutation in cancer of the cervix uteri and the vulva Int J Cancer 63 639 645 4 Horn D et al 1996 Three novel mutations of the NF1 gene detected by temperature gradient gel electrophoresis of exons 5 and 8 Electrophoresis 17 1559 1563 5 Wieland U et al 1996 Quantification of HIV 1 proviral DNA and analysis of genomic diversity b ypolymerase chain reaction and temperature gradient gel electrophoresis J Virology Methods 57 127 139 6 Kuhn J E et al 1995 Quantitation of human cytomegalovirus genomes in the brain of AIDS patients Journal of Medical Virology 47
51. de gel on gradient block A small volume of thermal coupling solution is applied to the gradient block eft panel The Polybond film with the polyacrylamide gel on top is put on the gradient block Slightly bend the Polybond film right panel in order to spread the thermal coupling solution evenly TGGE System 34 To position the polyacrylamide gel on the gradient block the Polybond film should be held between thumb and middle finger and slightly bended This leads to an even distribution of thermal coupling solution beneath the Polybond film If air bubbles are visible beneath the polyacrylamide gel try to squeeze them out by moving the gel slightly back and forth If this will not succeed completely remove the gel and repeat the aforementioned steps Don t touch the polyacrylamide gel directly with your fingers or your gloves Excess thermal coupling solution must be removed from the gradient block by using paper towels TGGE System 35 6 3 Perpendicular TGGE During perpendicular TGGE a mixture of molecules is separated over a wide temperature range The temperature gradient is perpendicular to the electrophoresis run direction Steps before TGGE sample preparation refer to chapter 4 1 programming of temperature gradient and electrophoresis parameters refer to chapter 3 2 2 Prepare in advance polyacrylamide gel attached to Polybond film running buffer 250 ml for each chamber pre cut and pre soaked
52. dependent change of conformation for separating molecules for review see Reference 1 Since the introduction of the first commercial available TGGE apparatus in 1989 temperature gradient gel electrophoresis has gained high interest in scientific and clinical laboratories due to the unprecedented resolution capability and easiness of analysis The range of scientific publications using the TGGE method is broad and covers all disciplines which use molecular biology methods e g Oncology Virology Immunology RNA Viroid Research Prion Research Population Analysis The TGGE method has also been used for quantitative analysis in indus try1 17 18 19 and for conformational analysis of proteins 1 2 Principle of method Conventional protein or nucleic acid electrophoresis separates molecules mainly according to size or charge TGGE adds a new parameter for separation namely the shape of the molecule The shape is mostly determined by the secondary and tertiary structure of the molecule and can be changed by external influences like temperature salt concentration pH etc The conformation both of proteins and nucleic acids depend on weak binding forces like hydrogen bonds or van der Waals bonds Increasing the temperature above a certain limit breaks down these bonds The molecules will adopt a so called denatured conformation in contrast to the native one E g with DNA it is possible to determine the temperature wh
53. diagram fig 21 In this diagram the Tm value is given on the y axis and the base pair number on the x axis The optimal fragment for TGGE analysis shows a stair type profile with 2 or 3 steps respectively fig 21 lower diagram The highest step the melting domain with the highest melting Temperature Tm is the artificial GC clamp Length and midpoint melting temperature of the 1 2 lower steps melting domains with lower Tms are determined by the original sequence of the DNA under study TernpPict diagram of optimized fragment Figure 21 Constructing the optimized DNA fragment TGGE System 67 When constructing an optimized fragment start with the TempPlot of an approximately 1000 bp sequence For PCR amplification select a fragment consisting of 1 2 melting domains Put the GC clamp at the more stable end or at any end if the fragment contains one melting domain Figure 22 schematically demonstrates how fragments with the utmost longest part of the sequence could be selected according to the TempPlot diagram The primers used for PCR amplification have to meet the following rules e Use non complementary primer sequences Do not allow base pairings of the last 3 bases at the 3 end neither with any other bases in the primer itself nor with the counterpart primer e Select primers 20 25 bp in length e Be sure that there are no additional primer annealing sites in the DNA sequence Favorable PCR products
54. dot matrix printer can be connected to the controller by using the port at the rear see figure 10 2 Choice whether a beep signal can be heard at the end of a program or when the program has reached an infinite time step 3 Choice between SA German or B English 4 not occupied 5 not occupied 6 not occupied TGGE System 19 Function key C Editing of programs program no __ Alist Bdel C quit D enter After entering a number of a non occupied program store the display shows Name gt lt ABCDEFGHIJKLMNOPQRST UVWXYZ a o lt gt amp A BABC C quit D enter L1 L6 alternative T1 T2 A BT1 Cqut D The temperature gradient between T1 and T2 must not exceed 45 K 1 L1 25 0 C L6G Alternative T1 T2 A Bdelete C quit D gt 1 L1 L6 Alternative T1 2 T2 A BLi Cqut D gt 1 L1 25 0 C L6 60 0 C T1 21 1 C T2 63 8 C Ok A Bno gt L1 Cquit D gt Or 1 L1 25 0 C L6 60 0 C T1 21 1 C T2 63 8 C Ok A Bno gt T1 Cquit Do In the main menu pressing C will offer the possibility to edit a new program First choose a program store number SA list displays all program stores from 0 to 99 with names or the information lt empty gt SB del deletes the last entry To give the program an individual name strike SB ABC to jump with the cursor into the let
55. duplices Aa and aA which have a non pairing base at the polymorphic site This non pairing base will lead to a shift of the Tm to lower values Tm3 and Ta Perpendicular TGGE shows at which temperature the different DNA strands will separate For future analysis by perpendicular or parallel TGGE a narrower temperature range which includes the Tm values of homo and heteroduplices can be used Please remember always that wild type and mutant DNA have to be mixed before PCR to get the 4 bands with different melting behavior Sometimes the melting difference between heteroduplices cannot be resolved but remember that 3 bands on the TGGE gel are enough to detect a mutation TGGE System 7 T1 cold T2 warm Gel slot Ting Heteroduplex aA Tm Homoduplex AA Ts Heteroduplex Aa Tm Homoduplex aa Figure 4 Schematic drawing of melting behavior of double stranded heterozygotic DNA with allele typ Aa after perpendicular TGGE Screening of multiple samples is performed by using parallel TGGE see above figure 2 Parallel TGGE looks like conventional SSCP analysis but has a higher probability to identify possible mutants T cold Twa Heteroduplex aA Tms Heteroduplex Aa Tmz Homoduplex aa Timi Homoduplex AA T gt warm Figure 5 Schematic drawing of a screening for double stranded heterozygotic DNA with allele type Aa after parallel TGGE Sometimes the melting difference between het
56. e solution under gentle vacuum for 3 5 min Water distilled fill up to 10 ml TEMED 22 5 ul 22 5 ul APS 4 Mix gently Avoid air bubbles Pour the gel solution into the glass plate sandwich immediately thereafter see chapter 4 1 2 without air bubbles Recipe for 10 ml gel solution 3 4 gels for Na TAE running buffer 3 Gel 5 Gel 8 Gel stock solution 30 0 8 40 w v ur 0 2x T Make sure that the urea has been completely resolved It is possible to heat up the urea containing solution slightly 40 C 50 C for a short time in order to improve the solubilization of urea De gas the solution under gentle vacuum for 3 5 min Water distilled fill up to 10 ml TEMED APS 4 Mix gently Avoid air bubbles Pour the gel solution into the glass plate sandwich immediately thereafter see chapter 4 1 2 without air bubbles TGGE System 28 Recipe for 10 ml gel solution 3 4 gels for ME MOPS EDTA running buffer 3 Gel E Gel 1 Gel Urea Cens 8 M stock solution 30 0 8 40 w v CEnd 1 X oie 40 Glycerol Cen 2 Water distilled Make sure that the urea has been completely resolved It is possible to heat up the urea containing solution slightly 40 C 50 C for a short time in order to improve the solubilization of urea De gas the solution under gentle vacuum for 3 5 min Water distilled fill up to 10 ml TEMED 1 1 APS 4 76 pl Mix gently A
57. e temperature gradient has to be reduced to 20 C 50 C 20 min running time necessary TGGE System 95 13 2 3 Electrophoresis parameters perpendicular gel figure 28 Sample volume 20ul Heteroduplex Hd 5ul load buffer 25ul run buffer Temperature range T1 T2 30 C 70 C Pre run 4 min 250 V 20 C Run 40 min 250 V 30 C 70 C parallel gel figure 29 Sample volume 2 ul sample 0 5 ul load buffer 2 5 u run buffer Temperature range T T2 40 C 60 C Pre run 4 min 300 V 25 C Run 10 min 300 V 40 C 60 C 13 3 Gel images This is what you should see on your gels if you proceed according to the protocol M Hd M M Mt Wt Hd Mt Wt Hd M Figure 28 perpendicular gel Figure 29 parallel gel M Marker Wt Wild type Mt Mutant Hd Heteroduplex TGGE System 96 14 Appendix 14 1 Technical Data 14 1 1_System Working temperature 4 C 35 C Humidity 10 80 14 1 2 Electrophoresis Chamber Temperature range of gradient block 5 C 80 C Maximum linear temperature range 45K for example 30 C 75 C above 20 C room temperature or 25 C 70 C Start of linear gradient parallel first marked line Start of linear gradient perpendicular end point of each line Temperature Accuracy 0 3 C Temperature Uniformity 0 3 C 2 mm Gradient block size 6cmx6cm Gel size W x L Run distance Size LxWxH Weight 7 4 cm x 8 0 cm 6 2 cm parallel 6 2
58. electrode wicks pre cut cover film cover glass plate treated on both sides with Acryl Glide e Because of the fixed orientation of the temperature gradient the removable electrophoresis chambers must be positioned as indicated in figure 14 Platform surrounding the gradient block 2 Electrophoresis buffer chamber with connector for cathode Gradient block 4 Electrophoresis buffer chamber with connector for anode Figure 14 Orientation of the two electrophoresis buffer chambers for perpendicular TGGE e Fill in the running buffer e g 0 1 x conc TBE into each electrophoresis chamber N Before you place the gel onto the gradient block be sure that the sample is ready for loading and cover film is available e To make full use of the linear range of the gradient block the polyacrylamide gel attached to the Polybond film should be positioned as indicated in figure 15 The rectangular and the marker slots of the gel are positioned at the beginning of the gradient block the marked lines represent the beginning of the linear range of the gradient block TGGE System 36 cs Pa en a ge ee ne en Sat a eee 7 BEER SE RE N RR RER Ree LE ELLE u Figure 15 Orientation of the polyacrylamide gel attached to the Polybond film on the gradient block during perpendicular TGGE See the position of the gel slots relative to the marked lines of the gradient block e T
59. eroduplices can not be resolved Different plates with pre fixed slots for 8 12 or 18 samples are available for screening purposes see chapters 6 4 and 15 TGGE System 8 2 General Recommendations 2 1 Safety warnings A gt A Check the voltage of the power supply and of the control unit before use In any case of malfunction of the power supply controller or electrophoresis chamber do not open the case but contact Biometra or your local distributor First switch off the power switch of the power supply before opening the lid of the electrophoresis chamber if you want to interrupt or stop the electrophoresis run During electrophoresis don t touch the electrode wires or the buffer inside the electrophoresis chamber High voltage Danger of life Whenever polyacrylamide gels are handled pay attention to standard laboratory safety regulations e g wear lab coat protective gloves and eye shield Polyacrylamide is neurotoxic 2 2 Notes for use e Do not scratch the protective foil of the gradient block In case of a damaged foil contact Biometra or your local distributor for a replacement foil e Do not use strong acids or basic solutions or organic solvents for cleaning glass plates the electrophoresis chamber or the gradient block e Do not incubate glass plates over night in cleaning solution e Wear protective gloves during all steps of the silver staining protocols to avoid staining artifacts due to the h
60. ersible transition into completely ssDNA the homoduplex bands become diffuse sometimes nearly invisible The sample loaded onto the gel only contains two different kinds of only homoduplices with nearly identical Tm The point mutation is located in one of the most stable melting domains parts of the fragment Only one species of DNA has been loaded onto the gel Wrong composition of buffer and or gel unstable or nonexistent temperature gradient No sigmoidal S shaped curve at all in the Run a perpendicular TGGE at first Estimate the effective temperature range of separation and the running time required for the DNA fragment to reach this range in a parallel TGGE run See 1 3 See Force heteroduplex formation by heating and reannealing the PCR sample prior to electrophoresis Calculate the melting map of your DNA fragment by the POLAND program Construct a new fragment which contains the site of mutation in one of the melting domains with the lowest Tm values See Theoretical backbone of a detection rate approximately 100 Check for the possibility that only one species of DNA was in your sample ineffective or nonexistent PCR amplification of a particular allele Loss of heterozygosity in a cell line etc Check all items listed under No S shaped curve in perpendicular TGGE TGGE System 88 corresponding perpendicular TGGE TGGE System 89 DNA fragment
61. es in DNA e RNA DNA e for RNA DNA hybrids in 1 M NaCl Sugimoto N Nakano S Katoh M Matsumura A Nakamuta H Ohmichi T Yoneyama M amp Sasaki M 1995 Biochemistry 34 11211 11216 Thermodynamic parameters to predict stability of RNA DNA hybrid duplexes e ENTROPY CORRECTION of base stacking Option not available by WWW DeltaS values of base stacking may be corrected by factors in order to simulate deviating ionic strengths The Delta S values of the thermodynamic parameters are multiplied with these factors The first is used for correction of all Delta S values the second only for A U stacks the third only for G C stacks Different values are used as defaults in dependence on the chosen thermodynamic parameter set e LOOP PARAMETERS thermodynamic parameters Option not available by WWW i e Sigma is fixed to 1 e 3 and loop entropy is calculated according to Poland Loops which appear during denaturation by internal base stack opening may be calculated by three different methods e p gt DeltaS loop SIGMA loop 1 1 75 according to Poland or Fixman amp Freire Use only with stacking parameters according to Porschke et al p p Gotoh p g or Klump p k e g gt DeltaS loop SIGMA DeltaS loop according to Gralla amp Crothers Use only with stacking parameters according to Turner et al p t e t gt DeltaS loop SIGMA DeltaS loop according to Turner
62. f the pH value of different electrophoresis buffers Buffer pH 20 C ApH AT 50 C 15 mM glycine NaOH 11 9 1 75 30 mM H3BO3 NaOH 10 0 0 52 40 mM Borax 20 mM H3BO3 9 1 0 44 25 mM glycine NaOH 9 4 1 66 30 mM Borax 75 mM H3BO3 8 6 0 33 89 mM Tris H3BO3 8 3 0 68 25 mM Tris glycine 8 3 1 05 375 mM Tris HCI 8 2 1 29 61 mM NaH2PO 10 mM NazgHPO 7 5 0 08 25 mM Na gt HPO 25 mM KH2PO 7 3 0 07 30 mM NaHPO 8 7 mM KH2PO 6 8 0 04 125 mM Tris HCI 6 8 1 39 23 mM NaHPO 132 mM NaH gt PO4 6 0 0 10 690 mM glycine 240 mM H3BO3 5 6 0 59 48 mM Tris H3PO4 5 5 0 18 48 mM KOH acetic acid 4 8 0 07 20 mM sodium acetate acetic acid 4 5 0 03 48 mM KOH acetic acid 3 6 0 09 690 mM glycine acetic acid 3 5 0 17 The pH value of 20 C is that with the optimum buffer capacity ApH is given for an incease in temperature of 50 C AT 50 C In the literature different buffer systems have been reported for the following proteins Dehydrogenases 41 B lactamase 1 tet repressor from E coli 33 42 alpha amylases 1 34 39 and serine proteases 40 TGGE System 80 12 Trouble shooting The following trouble shooting guide may be helpful in solving any problem that you may encounter If you need further assistance please do not hesitate to contact your local Biometra distributor or Biometra directly In any case where you recognize a failure which i
63. g a non denaturing polyacrylamide gel or a perpendicular TGGE gel Silver stain this gel Hot start PCR Adjust cycle parameters to finesse primer annealing Check on a perpendicular gel for ssDNA which does not show the sigmoidal inflection Change the PCR conditions in order to achieve even amplification of both single strands and force heteroduplex formation by heating and reannealing the PCR sample prior to electrophoresis Check the number of sigmoidal S shaped curves ona perpendicular TGGE Always use glass plates slot formers that are not damaged TGGE System 92 Parallel TGGE e unspecific PCR products of high molecular weight e ssDNA due to asymmetric PCR or ineffective renaturation Figure 27 More bands than expected TGGE System 93 13 TGGE Testkit 13 1 Introduction The TGGE Testkit contains samples for 4 parallel and 4 perpendicular TGGE runs with Na TAE buffer The samples contain a wild type mixture with one DNA double strand a mutant mixture with one DNA double strand and a heteroduplex mixture with four different DNA double strands two homoduplices and two heteroduplices Wild type min 20 ul Mutant min 20 ul Heteroduplex min 120 ul Loading buffer min 180 ul with blue marker for Na TAE buffer 13 2 Protocol 13 2 1 Gel composition 8 PAA 8M Urea 0 2x Na TAE 2 Glycerol Recipe for 10 ml gel solution 3 4 gels for Na TAE running buffer 8 Gel Urea ce
64. ger G 1994 Nucleic Acids Res 22 2760 2768 Thermal denaturation of double stranded nucleic acids prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction Original version of algorithm Poland D 1974 Biopolymers 13 1859 1871 Recursion relation generation of probability profiles for specific sequence macromolecules with long range correlations Fixman amp Freire 1977 Biopolymers 16 2693 2704 7 3 4 HELP for Poland Service 7 3 4 1 POLAND The program POLAND simulates transition curves of double stranded nucleic acids DNA and RNA as well as DNA RNA hybrids Additional information available OUTPUT RELATED PROGRAMS RESTRICTIONS ALGORITHM SUGGESTIONS PARAMETERS TGGE System 57 7 3 4 2 OUTPUT The program writes it output in numeric format which is converted to graphics by Tk Tcl Additional information available General description resolution 3Dplot GelPlot MeltPlot Temp50 Plot Resolution of graphics output The primary graphics output is produced as PostScript vector format That format is converted to GIF raster format this is a format directly displayed by your WWW browser The resolution of the GIF images is selectable 72 dots per inch 72 dpi is the standard screen resolution 150 dpi or 300 dpi are nice for printing But be aware on NanoWeak systems the higher resolutions need a lot of memory and tend to crash your system 3DPlot
65. he bands glass plate with 2 silicone smiles barriers 4 Wrong composition of 4 Check the composition of the gel gel and or and electrophoresis buffer electrophoresis buffer A salt front is in the gel During electrophoresis run this salt front is indicated by an abnormal mobility of the marker dyes The dye bands look extremely sharp sometimes the bromophenolblue band moves at the same position as the xylene cyanol blue band 5 High amounts of salt 5 Desalt the sample before loading ions in the sample Symptoms like described under 4 No S shaped curve in 1 Wrong buffer 1 Check the composition of the gel perpendicular TGGE fig 25 concentration ionic and the electrophoresis buffer strength in the gel is too high DNA has been denatured 2 Check the amount of urea added 2 Amount of urea in the to the gel The standard protocol gel is not sufficient The requires 8 M urea when dsDNA DNA has not been fragment GC contnent 55 75 denatured is analyzed on TGGE 3 Check the denaturation 3 Ineffective renaturation renaturation protocol used to form of the DNA only ssDNA heteroduplices has been loaded onto the gel 4 Consider adding a stabilizing 4 DNA fragment is not clamp to the fragment Evaluate stabilized by a GC rich the optimized DNA fragment for part of the sequence TGGE analysis by calculating the Irreversible melting in a melting pattern of the sequence one step transition into with the POL
66. ich is necessary to break down hydrogen bonds along double stranded DNA This temperature is called midpoint of transition Tm or melting temperature and characteristic for a certain stretch of DNA see figure 1 TGGE uses the melting temperature to identify DNA which differs in sequence among a mixture of molecules of the same size TGGE therefore not only separates molecules but gives additional information about the sequence DNA or RNA or the stability of proteins Migration branched DNA partially denatured conf single stranded DNA completely denatured double stranded DNA Temperature T cold Tu T warm Figure 1 Schematic drawing of different conformations of DNA during temperature gradient gel electrophoresis TGGE System 5 1 3 Special features of the TGGE System The microprocessor controlled gradient block of the TGGE System allows strictly defined linear gradients with high resolution A run distance of 2 mm which can easily be detected by eye corresponds to a maximum temperature difference of about 0 6 C Therefore even slightest differences of molecules can be detected by the new TGGE System Because of the small amount of material used for separation DNA or RNA fragments appear as fine bands which can clearly be distinguished from each other Even complex band patterns can be analyzed due to the high resolution capability of the gradient block Comparing the TGGE method with another screening method
67. igh sensitivity of the staining protocol TGGE System 3 Components ofthe TGGE System The TGGE System contains all components which are necessary to get started All kinds of TGGE applications parallel or perpendicular TGGE Constant Temperature GE Time resolved TGGE can be run with the System For certain applications which need different numbers or sizes of sample slots the adequate parts are available and can be ordered from Biometra or your local distributor see 6 3 Order Information The TGGE System Order number 024 000 consists of e TGGE Controller with integrated power supply 100 program 024 001 stores and control function of temperature and electrophoresis conditions e TGGE Electrophoresis unit with 2 removable buffer 024 002 chambers Peltier element powered gradient block and control cable e TGGE Starter Kit contains 024 003 3 plane Bonding glass plates 024 021 1 glass plate with 8 slots for parallel TGGE 024 022 1 glass plate with 1 rectangular slot for 024 023 perpendicular TGGE 1 glass pate with 12 slots for parallel TGGE 024 025 Electrophoresis wicks 100 pkg 024 015 Polybond film 25 pkg 024 030 1 Cover glass plate and 10 cover films 024 031 1 Acryl Glide 100 ml 211 319 e Manual When unpacking your System please check whether all mentioned parts are included If individual parts are missing call Biometra or your local distributor TGGE System 10 3 1 Electrophoresis unit
68. ilm pre cut 7 x 6 cm 25 pkg 024 032 TGGE Polybond film pre cut 8 8 x 8 8 cm 100 pkg 024 034 TGGE cover film pre cut 7 x 6 cm 100 pkg 024 035 TGGE Slotformer Slot forming units 10 x multi 9x long 024 121 Gel casting clips 3 pkg 010 007 Consumables Acrylamide bis Acrylamide 40 30 0 8 500 ml 210 254 Ammonium Persulfate 4x 25 g 210 486 EDTA Tetrasodium Salt Dihydrate 500 g 210 245 Glycerol 1 210 854 Sodium Acetate anhydrous 500 g 210 602 TEMED 25 ml 210 761 Acryl Glide 100 ml 211 319 Silver PAGE Silver staining kit for 20 stains 211 761 TRIS 1 kg 220 826 Consumables from Biometra are not available outside Germany Please source from another supplier TGGE System 106 17 Instructions for return shipment If you would like to send the unit back to us please read the following return instructions Should you have any problems with the TGGE System please contact your local Biometra dealer or our service department Biometra biomedizinische Analytik GmbH Service Department Rudolf Wissell Stra e 30 D 37079 Gottingen Phone 49 0 5 51 50 68 6 0 Fax 49 0 5 51 50 68 6 66 e Return only defective devices For technical problems which are not definitively recognisable as device faults please contact the Technical Service Department at Biometra e Use the original box or a similarly sturdy one e Label the outside of the box with CAUTION SENSITIVE INSTRUMENT e
69. l parallel TGGE applications can be run By simply switching the chambers perpendicular to the lines of the gradient block see figure 8 right panel it is possible to run perpendicular TGGE Figure 8 Orientation of the two electrophoresis buffer chambers represents cathode represents anode relative to the centrally positioned gradient block TGGE System 15 Programming L1 and L6 C aes vee ieee Bu aes ee seas eae Aa Table 1 Examples for actual temperatures on the gradient block programming L1 and L6 TGGE System 16 Programming T1 and T2 C Table 1 Examples for actual temperatures on the gradient block programming T and To TGGE System 17 3 2 Controller unit The controller is a highly integrated micro processor driven unit for controlling the temperature ramping time and ramping rate of the gradient block as well as supplying the power for the electrophoresis unit For entering and storing run parameters the front panel of the controller offers 4 function keys and a full numerical key pad During the run the display of the controller continuously shows the current parameters 3 2 1 Instrument keys and ports Power on off switch Display 4 function keys A B C D Alphanumerical key pad Computer port RS 232 Connectors for electrode cable red anode black cathode Printer
70. like SSCP shows superior performance of the TGGE method The controlled temperature conditions make repetition of experiments easy and lead to reproducible gel results The small format of the gradient block has been optimized in order to reduce sample volume and especially to save experimental time Perpendicular and parallel TGGE are two different modes applicable with the Biometra TGGE System without need for specialized parts or equipment Whereas perpendicular TGGE is mostly used for defining optimal separation conditions parallel TGGE allows the analysis of multiple samples e g screening perpendicular TGGE temperature gradient is perpendicular to the electrophoretic run direction one sample is spread over a broad temperature range parallel TGGE temperature gradient is parallel to run direction multiple samples are spread over a narrow temp range Perpendicular TGGE Parallel TGGE T cold T warm T cold ee Gel slots Run Ft direc tion Q T2 warm Figure 2 Schematic drawing of typical results after perpendicular TGGE left panel and parallel TGGE right panel TGGE System 6 1 4 How to start with TGGE analysis Starting with the gene of interest one develops the right combination of PCR primers for amplification of the desired gene fragment The polymorphic site must be located inside the amplicon and not at the far end The POLAND software helps one to identify suitable primers and
71. lips not correct to increase the pressure positioned Scratches on the 3 Use silicone grease along the spacers or old clips glass spacer but never on the sample slots Polybond film with gel can not Gel sticks to the glass 1 Glass plate with spacer must be easily removed from the plate with spacer treated with Acryl Glide before sandwich use Gel does not stick to the Gel has been poured 1 Pour the gel onto the hydrophilic Polybond film onto the hydrophobic side if you want to link the gel side of the Polybond covalently to the Polybond film film Hydrophobic face of the Polybond film must face the bonding plate Check the Polybond film with a drop of water for the hydrophilic and hydrophobic side Front top of gel shows a zig Gel has not been 1 Overlay the gel solution in the zag line overlaid with solution sandwich with 200ul Isopropyl or Isobutyl Alcohol Alternatively bi distilled water can be used Air bubbles in the gel The glass plate has not 1 Clean the glass plate and slot been cleaned carefully formers with ddH gt O and EtOH before use Avoid the intensive use of organic solvents They will dissolve the glue of the spacer and slot formers and thus remove them from the glass plate Sandwich was hold 2 Hold the sandwich at an angle of vertical during pouring 45 during pouring Solution poured to quick 3 Pour the gel solution slowly along or in the middle of the one side of the glass plate sandwich
72. llus polymyxa detected by temperature gradient gel electrophoresis Lessa E P and Applebaum G 1993 Screening techniques for detecting allelic variation in DNA sequences Molecular Ecology 2 119 129 Richter A Plobner L Schumacher J 1997 Quantitatives PCR Verfahren zur Bestimmung der Plasmidkopienzahl in rekombinanten Expressionssystemen BlOforum 20 545 547 Henco K and Heibey M 1990 Quantitative PCR the determination of template copy numbers by temperature gradient gel electrophoresis Nucleic Acids Research 18 6733 6734 Birmes A et al 1990 Analysis of the conformational transition of proteins by temperature gradient gel electrophoresis Electrophoresis 11 795 801 Arakawa T et al 1993 Analysis of the heat induced denaturation of proteins using temperature gradient gel electrophoresis Analytical Biochemistry 208 255 259 Chen X et al 1995 High resolution SSCP by optimization of the temperature by transverse TGGE Nucleic Acids Research 23 4524 4525 Scholz R B et al 1993 Rapid screening for Tp53 mutations by temperature gradient gel electrophoresis a comparison with SSCP analysis Human Molecular Genetics 2 2155 2158 Elphinstone and Baverstock P R 1997 Detecting mitochondrial genotypes by temperature gradient gel electrophoresis and heteroduplex analysis BioTechniques 23 982 986 Poland D 1974 Recursion relation generation of probability profiles for
73. m 175 422 432 Sanguinetti C J Neto E D and Simpson A J G 1994 BioTechniques 17 915 Kappes S Milde Langosch K Kressin P Passlack B Dockhorn Dwornczak B R hlke P and L ning T 1995 p53 Mutations in ovarian tumors detected by temperature gradient gel electrophoresis direct sequencing and immunohistochemistry Int J Cancer 64 52 59 Kluwe L MacCollin M Tatagiba M Thomas S Hazim W Haase W and Mautner V F 1998 Phenotypic variability associated with 14 splice site mutations in the NF2 gene American Journal of Medical Genetics 77 228 233 Lerman L S and Beldjord C 1998 Comprehensive mutation detection with denaturing gradient gel electrophoresis In R G H Cotton E Edkins and S Forrest eds Mutation Detection A Practical Approach Oxford University Press 35 62 Gamper H Piette J and Hearst J E 1984 Photochem Photobiol 40 29 ff TGGE System 105 16 Order information and spare parts Biometra offers a wide range of accessories parts and consumables related to Temperature Gradient Gel Electrophoresis Item Order No TGGE System 230 V electrophoresis unit with Peltier element powered gradient 024 000 block and 2 removable electrophoresis buffer chambers system controller with integrated power supply Starter Kit and manual TG
74. m 104 37 38 39 40 41 42 43 44 45 46 47 Zimmat R Gruner R Hecker R Steger G and Riesner D 1991 Analysis of mutations in viroid RNA by non denaturing and temperature gradient gel electrophoresis In R H Sarma and M H Sarma eds Structure amp Methods Vol 3 DNA amp RNA Adenine Press 339 357 Rosenbaum V Klahn T Lundberg Holmgren E von Gabain A and Riesner D 1992 Co existing structures of an mRNA stability determinat The 5 region of the Escherichia coli and Serratia marcescens ompA mRNA J Mol Biol in press Birmes A Sattler A Maurer S O and Riesner D 1990 Analysis of the conformational transitions of proteins by temperature gradient gel electrophoresis Electrophoresis 11 795 801 S ttler A Kanka S Schr rs W and Riesner D 1992 Random mutagenesis of the weak calcium binding side in SubtilisinCarlsberg and screening for thermal stability by temperature gradient gel electrophoresis Accepted for 1 International Symposium of Subtilisin Enzymes EMBL Hamburg Thatcher D and Hodson B 1931 Denaturation of proteins and nucleic acids by thermal gradient electrophoresis Biochem J 197 105 109 Wagenhofer M Hansen D and Hillen W 1988 Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studie by temperature gradient gel electrophoresis Analytical Bioche
75. m 8 M urea Using this same standard gradient in parallel TGGE is possible but time consuming since a longer running time is required to move the sample from the slot top of the gel to the effective range of separation middle or lower part of the gel where the domains begin to melt In other words much of the time consumed by electrophoresis is nonproductive since melting will not begin to occur until the DNA fragment has migrated a considerable distance Parallel TGGE can be easily be optimized by the information acquired from a preliminary perpendicular TGGE gel Figure 24 From perpendicular to parallel TGGE Steps 1 Determine the temperature range of effective separation This temperature interval is defined by two temperatures Tiow and Thigh If possible use two different DNA fragments one wild type and one mutated and perform heteroduplex analysis In this case Tiow is determined by the highest temperature where all duplices remain double stranded no retardation in the gel Thigh is determined by the melting temperature Tm of the most stable homoduplex see fig 24a In the event that only a wild type sequence is available determine Thigh by the melting temperature Tm of this sequence and define Tiow Thigh 10 C Note The temperature range of effective separation will have to be determined for each new DNA fragment analyzed 2 Using the information from step 1 select a temperature gradient for parallel TGGE
76. mated instrument called Blot Processor order number 015 000 or 015 090 Please contact Biometra or your local distributor to receive further information about the Blot Processor Wear non powdered protective gloves during all steps of the silver staining protocol to avoid staining artifacts due to the high sensitivity of the staining protocol e Remove the protective plastic sheets from the gel e Carefully remove any residual thermal coupling solution from the back of the gel Polybond film prior to staining e Put the polyacrylamide gel with the gel side upwards into the staining tray Avoid air bubbles during all staining steps e It s recommended to prepare at least 100 ml solution for each incubation step e If NaCl has been added to the gel running buffer incubate the TGGE gel for 15 min in Fixation solution to remove the NaCl TGGE System 45 Standard method Step Time Solutions Notes Fixation 5 min Fixation solution Silver Binding 10 min AgNO 3 Solution prepare freshly Washing 3x 1 min Fresh ddH20 demineralised water may be ok Developing 10 min Developer prepare freshly Stopping 5 min Stopping Solution Washing 10 min Rinse under fresh ddH2O Demineralised water may be ok Preparing for storage 1 5 h 50 glycerol Not absolutely necessary Preparing the gel for storage 1 5 h at room temperature in 50 glycerol is not necessary Staining sol
77. must be orientated to the Bonding glass plate On the hydrophilic side the polyacrylamide gel will polymerize and stick Press the hydrophobic side of the Polybond film firmly to the Bonding glass plate by using your thumb a rubber or a gel casting clip This will prevent that any polyacrylamide solution running between the Bonding glass plate and the Polybond film TGGE System 31 Glass plate sandwich e Assemble the Acryl Glide treated glass plate with spacer the Polybond film and the Bonding glass plate as indicated in figure 11 OOoogogogogo Figure 11 Setting up a gel sandwich for PAGE Bonding glass plate Polybond film and glass plate with spacer and slot former are assembled left panel The Polybond film is only visible at the inclined edge of the glass plate with spacer middle panel Fasten the clamps above the spacer to increase the pressure and to ensure a leakage free sandwich Figure 12 Pouring the polyacrylamide gel Initially 1 ml of polyacrylamide gel solution must be poured The gel sandwich must be hold at an angle of 45 when pouring left panel The solution must run along one side of the plate sandwich to avoid air bubbles The remaining 1 5 ml of solution is poured into the plate sandwich during which time the plate sandwich is slowly brought back into a vertical position right panel e Fill up the plate sandwich as high as possible The gel solution is overlayed with 200ul of Isop
78. n for high GC contents to avoid artificial hybrids e Renaturing at 50 C lower temperatures e g 37 C are applied for expected hybrids with multiple mismatches or for sequences with very low GC content The renaturation temperature should be approx 10 C below the Tm of the desired hybrid Samples should be dissolved in buffers with ionic strength identical to the ionic strength of the running buffer If samples are dissolved in buffer different to the running buffer the samples have to be equilibrated against the running buffer e g using dialysis Nucleic acids can be precipitated with ethanol and dissolved in denaturation renaturation buffer or running buffer TGGE System 30 5 4 Assembling the gel sandwich The thinness of the gel makes it necessary to cast polyacrylamide gels on a gel support film Polybond Order Number 024 030 Each sandwich consists of four elements Bonding glass plate without spacer Polybond film Polyacrylamide gel Glass plate with fixed spacer and fixed slot former different types of slots available Glass plates Glass plates must be dry and free of any dirt or dust Biometra recommends to wear powder free gloves even during cleaning of glass plates in order to prevent any skin debris which might interfere with silver staining Do not use strong acidic or basic solutions or organic solvents for cleaning the glass plates Do not incubate glass plates over night in cleaning solutions
79. n 8 M Acrylamide bis Acrylamide stock solution 30 0 8 40 w v 10x conc Na TAE pH 8 4 Cena 0 2 x conc 40 Glycerol Ceng 2 Water distilled Make sure that the urea has been completely resolved It is possible to heat up the urea containing solution slightly 40 C 50 C for a short time in order to improve the solubilization of urea De gas the solution under gentle vacuum for 3 5 min Water distilled fill up to 10 ml TEMED APS 4 Mix gently Avoid air bubbles Pour the gel solution into the glass plate sandwich immediately thereafter without air bubbles TGGE System 94 13 2 2 Running buffer 0 2x Na TAE Buffer composition Na TAE pH 8 4 1 M Sodium acetate Stock solution 10 x conc 10 mM EDTA 400 mM TRIS pH 8 4 titrate with Acetic Acid never use HCI Na TAE Running Buffer 0 2x conc Na TAE pH 8 4 TBE buffer not recommended for the TGGE Testkit Using TBE as running buffer results in less sharp bands longer running time and lower melting temperatures Perpendicular gel pre run 4 min run 40 min Using the 30 C 70 C temperature gradient allows no optimization of parameters for parallel TGGE as the melting curve starts just before the DNA is completely molten into single strands Parallel gel pre run 4 min run 10 min Using the 40 C 60 C temperature gradient gives no resolution as all double strands are molten and only single strands exist Th
80. naturation buffer to the sample and mix Heat at 95 C for 5 minutes denaturation Incubate at 50 C for 15 minutes renaturation The sample is then loaded directly to the gel In order to achieve the recommended loading volumes for diagonal or perpendicular TGGE refer to chapter 4 2 the samples should be filled up with running or loading buffer e Renaturing at 50 C higher temperatures higher stringency can be chosen for high GC contents to avoid artificial hybrids lower temperatures e g 37 C are applied for expected hybrids with multiple mismatches or for sequences with very low GC content The renaturation temperature should be approx 10 C below the Tm of the desired hybrid TGGE System 25 4 2 Quantity and volumes of samples Depending on the slot size of the gel Biometra recommends the following amounts of material glass plate with 1 rectangular slot 50 ul volume approx 50 ng DNA RNA of interest 2 marker slots 5 ul volume 3 5 ng DNA RNA of interest glass plate with 8 slots 5 ul volume 3 5 ng DNA RNA of interest glass plate with 12 slots 3 ul volume 1 3 ng DNA RNA of interest glass plate with 18 slots 2 ul volume approx 1 ng DNA RNA of interest If less volumes fill up the slots with running buffer or loading buffer to the volumes listed on top This will create better results 5 Setting up polyacrylamide gels 5 1 Selecting Concentration of PAA gels The TGGE System represe
81. ng nearest neighbor stacking interactions and loop entropy functions described in the literature An extension of the algorithm the virtual stack model allows for the incorporation of specific mismatched sequence positions in the stability calculations as described by Heinz Werntges The input data required for calculation are e the sequence lt 1 000 bp Use GCG format or plain format without spacing Plain format accepts only 180 characters per line e mismatched positions optional e the strand concentration affecting the dissociation temperature use the programmed standard e parameter set selection DNA DNA low salt RNA oligo long ds e output format options choose GIF format Data sets predicted by the program comprise the following e A perspective view on the temperature dependent denaturation profile that is denaturation probability vs sequence position vs temperature e The temperature dependent relative uv hypochromicities as they would be measured in optical melting at wavelengths of 260 and 280 nm 282 nm in case of DNA respectively full hypochromicity corresponds to approx 30 of the OD at low temperature e The derivative form of above hypochromicities showing the melting temperature s and corresponding half width s of the transitions s giving hints about the transition cooperativity TGGE System 52 e Predicted relative gel mobility as calculated according to Lerman et al as a graph
82. ntaminated YES NO delete Method of cleaning decontamination NAME POSITION HEAD OF DIV DEP INSTITUTE COMPANY SIGNED DATE PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER TGGE System 108 19 Warranty This Biometra instrument has been carefully built inspected and quality controlled before dispatch Hereby Biometra warrants that this instrument conforms to the specifications given in this manual This warranty covers defects in materials or workmanship for 12 month as described under the following conditions This warranty is valid for 12 month from date of shipment to the customer from Biometra or an authorized distributor This warranty will not be extended to a third party without a written agreement of Biometra This warranty covers only the instrument and all original accessories delivered with the instrument This warranty is valid only if the instrument is operated as described in the manual Biometra will repair or replace each part which is returned and found to be defective This warranty does not apply to wear from normal use failure to follow operating instructions negligence or to parts altered or abused
83. nteractions and loop entropy functions described in the literature The input data required for calculation are the sequence of course and no default here optional mismatched positions the strand concentration affecting the dissociation temperature the method to calculate the final dissociation into single strands the thermodynamic parameter set DNA DNA low salt RNA and the temperature range in which the calculation is performed In case you need access to the full range of input options more options are available to the experts Data sets predicted and figures drawn by the program are described below see also for OUTPUT e A perspective view on the temperature dependent denaturation profile denaturation probability vs sequence position vs temperature This plot does not include the dissociation of dsNA into single strands thus it shows most clearly the relative stability of the different parts of the NA e The temperature dependent relative UV hypochromicities as measured in optical melting at wavelengths of 260 and 280 nm 282 nm in case of DNA respectively full hypochromicity corresponds to approx 30 of the OD at low temperature e The derivative form of above hypochromicities showing the melting temperature s and corresponding half width s of the transitions s giving hints about the transition cooperativity TGGE System 56 e Predicted relative gel mobility calculated according to Lerman et al vs
84. nts a highly optimized system for performing flat bed polyacrylamide gel electrophoresis under defined temperature conditions In addition to typical TGGE applications the system is ideally suited to run standard fragment separations without temperature gradient very quickly Depending on the molecular weight of the sample we recommend the following acrylamide bisacrylamide concentrations Conc DNA fragment length 3 gt 1000 bp 5 500 1000 bp 8 lt 500 pb 5 2 Setting up the gel solution Each gel sandwich contains approx 2 5 ml polyacrylamide solution We therefore recommend to prepare 10 ml solution to pour 3 4 gels at the same time Polymerized gels which are not immediately used must be stored at room temperature To inhibit any gel drying we recommend to wrap the polymerized gels into saran foil or wet plastic bags Wet towels can be used only for short time storage TGGE System 26 Keep in mind that polymerized polyacrylamide gels which include urea should not be used after 2 4 days of storage depending on storage conditions TGGE System 27 Recipe for 10 ml gel solution 3 4 gels for TBE running buffer 3 Gel 5 Gel 8 Gel stock solution 30 0 8 40 w v CEnd 0 1 x Conc Make sure that the urea has been completely resolved It is possible to heat up the urea containing solution slightly 40 C 50 C for a short time in order to improve the solubilization of urea De gas th
85. o gt T1 leads back to programming T1 Pressing BB L1 allows programming of L1 and L6 Depending on the decision of programming L1 and L6 or T1 and T2 the programmed temperatures will be shown in the display After programming L1 and L6 this temperatures will be shown in the display during the next programming steps TGGE System 20 1 11 25 0 C L6 60 0 C time __ Om Os El OV 500mA 30W A BV h Caquit D gt 1 L1 25 0 C L6 60 0 C time 30m Os El OV 500mA 30W A BV h Cquit D gt 1 L1 L6 V h __0 00 Vh El OV 500mA 30W A BTime Cquit D gt 1 11 25 0 C L6 60 0 C time 30m Os Ek _ OV 500mA 30W A B special C quit D gt 1 L1 25 0 C L6 60 0 C time 30m Os El 250V 500mA 30W A B special C quit D gt 1 special functions ramptime _ 0m Os A B standard C quit D gt 1 L1 25 0 C L6 60 0 C Time 30m Os El 250V 500mA 30W A B special C quit D gt 2 L1 __ L2 alternative T1 T2 A BT1 C quit D program no pgm end _ step s runtime NM S L1 22 0 C L6 22 0 C block off A B Elpho C programs D Time You can choose electrophoresis time Standard values for a TGGE run are 30 45 min If you enter 30 and confirm by press ing SD enter you get 30 s If you enter 30 you will get 30 min If you enter 30 you will get 30 h V h Pressing SB V h replaces times by
86. ol and 100 ml acetic acid are adjusted with double distilled water to 1 liter 30 EtOH Prepare freshly 60 ml ethanol in 140 ml double distilled water Prepare Silver Binding Agent by reconstituting contents of one pouch in 1 of ddH 0 This solution must be prepared fresh every time Immediately before staining add 0 7 ml of 37 Formaldehyde to 200 ml of reconstituted Silver Binding Agent Just prior to use prepare developing solution by reconstituting contents of one pouch of Developer and 15 mg of Developer II in 200 ml of ddH 0 This solution must be prepared fresh every time Immediately before developing add 0 7 ml of 37 Formaldehyde to 200 ml of reconstituted developing solution 7 5 Acetic Acid 75 ml acetic acid are adjusted with double distilled water to 1 I TGGE System 48 6 6 Ethidium bromide staining Incubate the gel in staining solution 0 5 ug ml ethidium bromide in 1 x conc TBE for 30 45 min Analyze under UV radiation 27 6 7 Blotting DNA from TGGE gels can be blotted onto a solid state support either by electroblotting Fastblot or vacuum blotting Vacu Blot System If DNA is to be blotted after TGGE analysis the TGGE gel must be poured onto the hydrophobic side of the gel support film Polybond film Otherwise the gel cannot be detached from the gel support film 6 8 Autoradiography TGGE gels can also be directly exposed to x ray films is radiolabeled samples are analyzed Direc
87. ore trouble to support that machine Now make a decision NEW Server OLD Server Poland request form Foland request torm u eh su Poland expert request form Institut f r Physikalische Biologie Department of Biophysics Heinrich Heine Universit t D sseldorf Germany Feb 26 1999 G Steger M Labensky A J ger TGGE System 51 7 2 The old Poland program Old Server 7 2 1 About the Poland service The Poland program is an experimental service of the University of D sseldorf Biophysics Department and thus the whole set up access and service are subject to change The Poland server will calculate thermal denaturation profiles and temperature dependent uv absorbance or gel mobility of double stranded RNA or DNA based on sequence input and parameter settings in the request form Details below The program used in these calculations was developed by Gerhard Steger for comparing theoretical predictions to experimental data mainly optical denaturation profiles taken at 260 and 280 nm and TGGE temperature gradient gel electrophoresis experiments The original version was written in VAX Fortran VMS using the Graphics Kernel System GKS for data presentation 7 2 2 Program specific information Calculation is based on D Poland s algorithm in the implementation described by Gerhard Steger The Poland algorithm calculates the denaturation profile for double stranded nucleic acid usi
88. port Interface to electrophoresis unit VRP RADY Mains and fuses Figure 10 TGGE System Controller from the rear TGGE System 18 3 2 2 Programming of the TGGE Controller After switching on the controller the display shows the instruments name and the software version Immediately afterwards the main menu appears Main menu T1 22 0 C T2 22 0 C block off A BElpho C programs D Function key D Options 1 print programs 2 signal 3 language At BJ Caquit D enter 4 standard mode 5 test mode 6 void At B Caquit D enter WA and B 4 allow scrolling of display By C quit you will return to the main menu At the bottom line of the display 4 possible options which can be retrieved by the 4 functions keys SA B C D are shown These 4 options change during programming relative to the chosen menu Temperatures T1 and T2 shown in the display are dependent on the room temperature BA comments or tips about the current program step SB Elpho commands to load and start the program SC programs commands to edit new programs change or de lete existing programs different options like printing of program stores or of running protocols choice of language choice of signal D In general select by scrolling activate by pressing enter except when selecting program numbers or temperature values Printing of program stores A
89. r Sigma l p internal loops according to Poland a f algorithm according to Fixman amp Freire t 90 120 0 5 Temperature range and steps DNA Thermodynamic values according to Gotoh et al and Klump both are ideally suited for calculations The parameter set of Breslauer et al does not fit our experiments The parameter set of Allawi amp SantaLucia is based on a reevaluation of all known parameter sets for DNA i e this set may the optimal one For references to the original thermodynamic parameter sets see here Additional information available Gotoh Breslauer et al Klump SantaLucia et al Allawi amp SantaLucia e Gotoh Optimal parameter combination for Gotoh d 1 000 DeltaS factor n 1 e 3 Dissociation constant B c 1 e 6 cO 1E 9 to 1E 11 s 1 e 3 loop parameter Sigma l p internal loops according to Poland a f algorithm according to Fixman amp Freire t 60 80 0 5 Temperature range and steps e Breslauer et al No optimal parameter combination for Breslauer et al e Klump Optimal parameter combination for Klump t 70 90 0 5 d 1 000 DeltaS factor n 1 e 3 Dissociation constant B c 1 e 6 cO 1E 9 to 1E 11 s 1 e 3 loop parameter Sigma l p internal loops according to Poland a f algorithm according to Fixman amp Freire Temperature range and steps TGGE System 60 e SantaLucia etal e Allawi amp SantaLucia This is
90. r fragment will form the hairpin and both will have the same mobility Only at temperatures within the range of Tiow T high will the base substituted DNA be distinguishable from the wild type DNA TGGE System 73 By virtue of the temperature gradient which TGGE imposes upon the gel one particular area of the gel will provide the appropriate temperature range i e Tiow Thigh to allow formation of the hairpin and hence visualization of differences in mobility 9 2 DNA sample preparation Add 3 ul of 95 formamide 10 mM EDTA to a 3ul aliquot of the PCR amplified 9 3 Gel casting Recipe for 10 ml gel solution 3 4 gels for SSCP ME running buffer 3 Gel 5 Gel 8 Gel Stock solution 30 0 8 40 w v CEnd 1 x Conc 40 Glycerol cena 2 Water distilled Make sure that the urea has been completely resolved It is possible to heat up the urea containing solution slightly 40 C 50 C for a short time in order to improve the solubilization of urea De gas the solution under gentle vacuum for 3 5 min Water distilled fill up to 10 ml TEMED Mix gently Avoid air bubbles Pour the gel solution into the glass plate sandwich immediately thereafter see chapter 4 1 2 without air bubbles Cast the TGGE gels according to the instructions given under Setting up polyacrylamide gels TGGE System 74 TGGE System 75 9 4 Electrophoresis Perpendicular TGGE with a 12 or 18 slot gel
91. r very GC rich sequences raise the temperature in the TGGE gel 40 C 80 C or lower the ionic strength in the electrophoresis buffer and gel by using another buffering system electrophoresis buffer 0 1 x conc TBE 8 9 mM Tris 8 9 mM boric acid 0 24 mM EDTA gel 0 1 x conc TBE buffer 5 polyacrylamide 8 M urea temperature gradient 35 60 C published for analysis of dsSCARNAS 1 33 34 36 37 and reovirus RNA 34 Note The reduction in ionic strength will lower the Tm 10 2 Partly double stranded RNA e g viroid RNA Use the suggested protocols provided in this manual for ME buffer or refer to the buffer and gel systems published in various papers electrophoresis buffer 0 2 x conc TBE 17 8 mM Tris 17 8 mM boric acid 0 4 mM EDTA gel 0 2 x conc TBE 5 polyacrylamide no urea 1 32 33 34 37 10 3 Single stranded RNA with single hairpin structures m RNA secondary structures Use standard protocols given in this manual for ME buffer or refer to other buffer and gel systems described in the literature 38 electrophoresis buffer 10 mM sodium phosphate pH 6 0 with or without 1 mM MgCl gel 8 polyacrylamide 10 mM sodium phosphate pH 6 0 with or without 1 mM MgClz TGGE System 77 10 4 Staining For detection of the RNA silver staining is recommended For identification of double stranded virus RNA from crude plant extracts a protocol based on immunoblotting has been published 35 TGGE
92. ropyl Alcohol 2 Propanol Isopropanol or Isobutyl Alcohol 2 Methyl 1 propanol Isobutanol or distilled water to produce a horizontal surface of the gel TGGE System 32 e Polymerization of the gel must be at least for 0 5 h better for 1 1 5 h or optional over night at room temperature The sandwich should stand up vertically and must not be moved during polymerization e Gels may be stored up to 4 days at room temperature wrapped in wet paper towels in a plastic bag Do not store at 4 C 5 5 Disassembling the gel sandwich e Remove the clamps from the plate sandwich e Remove the Bonding glass plate from the sandwich by sliding it smoothly The gel polymerized to the Polybond film will adhere to the other glass plate e Withdraw the Polybond film with the adhering polyacrylamide gel carefully from the other glass plate In the area of the slot former remove the Polybond film very carefully to avoid any damage to the slots e If slots show distortion or wrinkles don t fill in samples because after electrophoresis bands in this lane will show distortion as well TGGE System 33 6 Electrophoresis with the TGGE System The electrophoresis unit of the TGGE System has been designed to accommodate all TGGE and related applications like CTGE TTGE and SSCP without cumbersome changes It s easy to switch between perpendicular parallel or diagonal TGGE for adequate accessories see chapter 6 4 6 1 Electrophoresis conditions
93. s have not reached the effective range of separation Temperature at the cathode side of the gel is too high The DNA enters the gel as completely ssDNA T T Tdiss low high Figure 26 a No heteroduplex bands in parallel TGGE TGGE System 90 Corresponding perpendicular TGGE 20C leoc Tm values of homoduplexes are nearly identical Tm1 Tm2 R Tm2 Tm1 lt 0 1 C Tm2 Tm1 Tm2 un 20 C__ __ 6o c point mutation is located in the most stable part of the sequence By for these or similar patterns see No S sha curve in perpendicular TGGE Figure 26 b No heteroduplex bands in parallel TGGE TGGE System 91 Problem Cause Solutions Gel visualizes more bands than expected fig 27 PCR artifacts Nonspecific high molecular weight products are sometimes obtained by PCR amplification of genomic DNA samples Due to the extremely sensitive silver staining they become visible on a TGGE gel although the PCR product seemed to be clean on an agarose gel stained by ethidium bromide ssDNA due to asymmetric PCR or ineffective renaturation during the formation of heteroduplices is visualized on the gel by a band of orange to brown red color DNA simply contains more species than expected The slot formers on the glass plates are damaged Ghost bands are caused by these imperfect slot formers Recheck the sample by runnin
94. s marked in the list by an exclamation mark please stop working with the instrument and call the local representative for replacing faulty parts Problem Cause Solutions Preparing the gel solution Urea can not be dissolved in gel solution Dissolving urea is an endothermic process and requires energy in form of heat Heat up the acrylamide urea solution but not more than 40 C 50 C Mix the solution Gel does not polymerize Old chemicals Prepare acrylamide bis acrylamide solution freshly Prepare 4 APS freshly and freeze in small aliquots Gel solution prepared 2 Check all reagents that have been incorrectly included in the gel solution and mix thoroughly To much oxygen inthe 3 Degas solution before adding gel solution TEMED and APS Gel polymerizes to fast To much TEMED and 1 Check the concentrations of APS has been added to TEMED and APS Use the the gel solution amounts given in the standard protocol The gel solution has 2 Allow the gel solution to cool been heated in order to dissolve the urea down to room temperature before adding TEMED and APS Note the gel solution should not be warmed up to more than 50 C TGGE System 81 Preparing the gel sandwich Gel solution leaks out of Gel sandwich has been 1 Clean spacers with methanol sandwich set up incorrectly 2 Fasten the clips above the spacer C
95. sequence specific macromolecules with long range correlations Biopolymers 13 1859 1871 Lerman L S and Silverstein K 1987 Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis Meth Enzymol 155 482 501 Steger G 1994 Thermal denaturation of double stranded nucleic acids prediction of temperatures critical for gradient electrophoresis and polymerase chain reaction TGGE System 103 26 27 28 29 30 31 32 33 34 35 36 Schumacher J Randels J W and Riesner D 1983 A two dimensional electrophoretic technique for detection of circular viroids and virusoids Anal Biochem 135 288 295 Sambrook J Fritsch E F and Maniatis T 1989 Molecular cloning Cold Spring Habor Laboratory press Steger G and Riesner D 1992 Temperaturgradienten Gelelektrophorese eine Methode zur Analyse von Konformationsubergangen und Mutationen in Nukleins uren und Proteinen In Radola B J ed Handbuch der elektrophorese VCH Verlagsgesellschaft Weinheim Sheffield V C Cox D R and Lerman R M 1989 Attachment of a 40 base pair G C rich sequence GC clamp to genomic DNA fragments by the polymerase chain reactiob results in improved detection of single base changes Proc Natl Acad Sci USA 86 232 236 Hecker R Wang Z Steger G and Riesner D 1988 Analysis of RNA structure by temperature gradient gel electrophoresis viroi
96. soralen Furo 3 2 g coumarin C141H603 68 7 4 3 POLAND analysis Of samples 22222222snnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 69 8 Optimizing parallel TGGE by perpendicular TGGE cccccccceeeeeeeeeeeees 70 8 1 Check short DNA fragments for their melting behavior 70 8 2 From perpendicular to parallel TGGE cccccccseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 71 9 ATGGELSSCHR nee ee 72 9 1 Running an SSCP on the TEBGE ars eeen 72 9 2 DNA sample preparation ccccccccccsessseeeeeeeeeeeeeeeeeseeeeeeeeeeeseeeeesseeeeeeess 73 93 ASSL re ALLO E cas Aa Ses A aes dae cas deh does dae cas dae doa deh ce 73 GA TEISGIOPMOGE SIS ernen eens cence E e teenies E Ee eee aie ip EEEE EERE Er 75 9 5 Rouline analysis ss222 542 RER RER eee eee eee 75 10 TSGE i RNA analysis ara ee ei ae 76 10 1 Completely double stranded RNA uuuussssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 76 10 2 Partly double stranded RNA e g viroid RNA eeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 76 1053 22052 Single stranded RNA with single hairpin structures m RNA secondary SITLIELUF S Hr A errechnen 76 19 4 Stang are Sr ae ee ee seele ueber 77 11 TGGE in protein analysis 2 2 2 reece ree rae ree awa at 78 sto it BUES 2a rotor ele sorutinte Preven Peter te Pre rt ee ehe 78 12 Trouble sho0ting 22 25 22 2 22 22 conesececcenacas scwesececcenacae scwesececcenacaccceeseeuce
97. t exposure Incubate the TGGE gel for 15 min in Fixation solution see 6 5 Silver staining Optional Silver stain the gel Remove residual buffer from the gel Expose to an x ray film at room temperature Exposure of dried TGGE gels Incubate the TGGE gel for 15 min in Fixation solution see 6 5 Silver staining Optional Silver stain the gel Incubate the gel in 2 5 glycerol for 10 minutes to prevent the gel from cracking Incubate cellophane on the gel Air dry at room temperature for one day or use a geldryer at 50 C for at least 3h Exposure to an X ray film 6 9 Elution of DNA from the TGGE gel DNA fragments which have been separated on TGGE for example different alleles of one gene can be eluted from silver stained TGGE gel and reamplified by PCR Using a Pasteur pipet punture the gel and extract a ul piece containing the particular DNA duplex Incubate in 20 ul TE buffer overnight Use a 1 ul aliquot for reamplification TGGE System 49 7 TGGE in analysis of point mutations in dsDNA For analysis of point mutations in dsDNA an extremely high detection rate of greater than 95 is routinely achieved when the experiment is carefully planned The next two chapters provide information for optimizing detection of base substitutions 7 1 Theoretical background of a detection rate approximating 100 for point mutations calculations with the POLAND program DNA does not melt by deannealing base pair by base pair from
98. temperature for different values of the retardation length parameter Lr This plot can be used for direct comparison with TGGE experiments superpositions of plots generated with or without mismatched positions given are useful as a hint whether specific mismatched duplexes could be detected among homoduplexed DNA in a mixture of sample and reference double strands having undergone a denaturation renaturation cycle using either perpendicular or parallel TGGE e A half denaturation temperature plot showing the temperature at which each base pair has a probability of 50 to be in the open state Similar to the three dimensional plot this plot can be used to estimate the destabilizing effect of mismatches on the surrounding part of the sequence a temperature shift of the TGGE transition can be expected if the lowest melting part of the sequence is directly affected by the mismatch Calculations can be done for oligonucleotides gt 15 bases or long double strands gt 50 bases respectively In the case of oligonucleotide mode a length dependent correction for the strand dissociation process is applied We do not have sufficient experimental results to stringently check for this mode to give valid results but for the length range of about 20 nucleotides there is at least experimental evidence Using oligo mode with far longer sequences gives misleading results 7 3 3 References for Poland Service Description of implemented programs Ste
99. ter field Moving inside this field is possible by the keys SA gt and B lt Pressing SD enter the current high lighted letter is stored in the name field This step can be repeated 8x times Pressing D enter two times leads to the next step It is now possible to enter for the first program step temperature values for both sides of the gradient block Alternatively the temperatures for L1 and L6 first and last thick lane on the gradient block or for T1 and T2 left and right edge of the gradient block can be programmed The change between programming L and T can be done by pressing B T1 If a number has been entered at the field L1 you have to confirm by pressing D enter The cursor jumps to L6 After programming L6 you will be asked ok Pressing B no L1 allows new programming of L1 and L2 Pressing SD yes confirms the temperatures After entering a temperature for T1 T2 L1 or L6 the temperature can be Deleted by pressing B delete If programming of T1 and T2 left and right edge of the gradient block has been done but L1 and L6 are preferred changing to L1 and L6 can be done by pressing B L1 After entering L6 or T2 all four temperatures L1 L6 T1 T2 are displayed ok Pressing SD yes leads to the programming of the Electrophoresis parameters Pressing SB no gt L1 leads back to programming L1 Pressing SB T1 allows programming of T1 and T2 Pressing B n
100. the unified parameter set RNA DNA Thermodynamic values according to Sugimoto et al 1995 in 1 M NaCl The top strand is RNA the bottom strand is DNA 5 r 3 3 d 5 the input sequence is the RNA strand lonic strength dependence Following values may be used for correction of calculated Tm values Tm 2 Tm 1 G C I G C 1 f G C K A U log c2 c1 with Tm transition midpoint melting temperature c ionic strength concentration of Na ions f G C G C content I X Y dependence of ionic strength of base pair type X Y DNA I A T 18 3 C Owen Hill amp Lapage 1969 Biopolymers 7 503 516 G C 11 3 C Frank Kamenetskii 1971 Biopolymers 10 2623 2624 RNA I A U 20 0 C Steger Muller amp Riesner 1980 G C 8 4 C Biochim Biophys Acta 606 274 284 TGGE System 61 7 3 4 7 PARAMETERS Additional information available BASE_STACKING _ thermodynamic_parameters ENTROPY_CORRECTION_of_base_stacking LOOP_PARAMETERS _ thermodynamic_parameters TEMPERATURE_RANGE_OF_CALCULATION MISMATCHED_POSITIONS_in_original_sequence CONCENTRATION_and_DISSOCIATION_CONSTANT STIFFNESS_of_nucleic_acid THERMODYNAMIC PARAMETER SETS for BASE STACKING You can select between five different thermodynamic parameter sets of base stacking for loop parameters see below Additional information available RNA DNA RNA DNA e RNA e for RNA in 1M NaCl Freier S M Kierzek R Jaeger
101. ties with interpretation of band pattern melting profile etc For example in parallel TGGE nonspecific bands with a higher molecular weight than the specific PCR product may be misinterpreted as heteroduplices or analogs with lower thermal stabilities Therefore before running a TGGE gel please check the PCR product and if necessary purify the specific PCR product of interest e g by agarose gel electrophoresis and subsequent gel extraction Sample preparation for direct DNA analysis 1 volume of DNA RNA samples are dissolved with 1 volume of TBE or Na TAE loading buffer or with 0 1 volume of the total loading volume ME loading buffer see Appendix 2 The resulting mixture is loaded directly on to the polyacrylamide gels Secure that the slots are filled up to maximum if necessary add loading buffer to fill up the slots to maximum In case of low concentration samples we recommend to prepare 5x conc loading buffer 0 2 volume of this concentrated loading buffer is mixed with the sample and loaded onto the polyacrylamide gel Denaturation Renaturation for heteroduplex analysis of DNA For heteroduplex analysis the samples are denatured and renatured prior to TGGE Quantitative denaturation is accomplished by heating in 4 M urea The following protocol is recommended for all DNA fragments with GC contents of 50 70 Depending on the buffer to be used for electrophoresis add one sample volume of corresponding DR buffer denaturation re
102. til the samples have fully entered the polyacrylamide gel unlike with the former TGGE System of QIAGEN this process will only take 1 3 minutes and have moved about 3 5 mm in the gel Stop the electrophoresis run open the safety lid Rinse the now empty slots with 0 5 1 ml running buffer Cover the polyacrylamide gel including the slots with the 7 x 6 cm pre cut cover film see figure 17 A small buffer layer must remain between cover foil and gel Avoid air bubbles The cover film must be positioned with the long side parallel to the VAN buffer chambers perpendicular to the arrow on the safety lid Soak any excessive buffer from the side of the gel The gel must not swim in buffer solution TGGE System 42 Figure 17 The polyacrylamide gel has to be covered by a pre cut hydrophobic cover film A small buffer layer remains between gel and cover film Polybond film wicks slot of polyacrylamide gel e Bring the electrode wicks to an overlap with the cover film The overlap between wick and cover film should be almost 2 cm see figure 17 Avoid air bubbles e Be sure that the 2 silicone barriers are fixed to the cover glass plate before use e Cover the sandwich with the Acryl Glide treated cover glass plate see figure 18 The silicone barriers have to be positioned perpendicular to the wicks and never on top of the wicks Figure 18 Side view of the polyacrylamide gel 8 on top of
103. utions Fixation Silver Binding Developing Solution Stopping Solution 10 0 5 EtOH Acetic Acid 100 ml ethanol and 5 ml acetic acid are adjusted with distilled water to 1 liter 0 19 AgNO 1 9g AgNO is dissolved in 1 liter of distilled water Can be reused 5 times Store dark 1 5 0 08 0 1 NaOH NaBH Formaldehyde Dissolve 15 g NaOH in 1 liter distilled water Add 0 89 NaBH and 2 7 ml formaldehyde stock solution 37 in water This buffer must be freshly prepared immediately before use 0 75 Na CO3 Dissolve 7 5 g sodium carbonate in ddH O Total volume 1 liter TGGE System 46 Quick method for PCR products Sanguinetti et al Step Time Solutions Notes Fixation 3 min Fixation solution prepare freshly Silver Binding 5 min AgNO Solution prepare freshly Washing 3x 1 min Fresh ddH20 Demineralised water may be ok Developing 5 min Developing solution prepare freshly Stopping 5 min Ethanol and acetic acid solution Washing 10 min Rinse under fresh ddH20 Demineralised water may be ok Preparing for 1 5 h 50 glycerol Not absolutely storage necessary Drying Room temperature Preparing the gel for storage 1 5 h at room temperature in 50 glycerol is not necessary Staining solutions Fixation 10 0 5 EtOH Glacial Acid Silver Binding Developing Solution Stopping Solution 100 ml ethanol
104. vice polandform html POLAND software predicts location and Tm values of melting domains for dsDNA and dsRNA as well as their perpendicular TGGE pattern The ability to predict the melting behavior of particular DNA fragments enables one to construct DNA fragments with optimized melting behavior resulting in a nearly 100 detection rate for point mutations inside of this fragment Since the end of February 1999 the POLAND program is available in two versions Using the above internet address allows the user to select between the old POLAND request form Standard the old POLAND expert request form or the new POLAND request form The following information is visible on the screen TGGE System 50 Poland Server ANNOUNCEMENT The WWW server for prediction of nucleic acid s thermal stability called Poland server according to the author of the basic mathematics will move during the near future to another computer This is not a mere relocation of the program that you have used up to now but the input output procedure is completely rewritten for the new server The new server produces better nicer plots and has a better more elongated help file But be aware of new bugs which might be introduced during the rewriting and relocation The old server both the standard and the expert form are unchanged Both forms will be available for the near future However that server is running on our DEC Alpha under OpenVMS and we run into more and m
105. void air bubbles Pour the gel solution into the glass plate sandwich immediately thereafter see chapter 4 1 2 without air bubbles 5 3 Some remarks corresponding to standard TGGE conditions Electrophoresis buffer running buffer e Always membrane filtrate e g 0 45um pore size the buffers before use e Running buffer always use the concentration identical with the gel condition e TBE is the most common used buffer system but the electrophoresis is not as fast as with Na TAE buffer It is possible to add up to 5 mM NaCl if a higher ionic strength is desired for reversible melting processes which are required for parallel TGGE in multiple sample analysis A higher NaCl concentration should not be used because it causes an unacceptable high electrical current e Na TAE is the buffer for fastest electrophoresis e ME buffer meets all the requirements of a variety of TBE buffers with different ionic strengths but is only stable for a very short time Stable for about 3 days Do not use as the buffer becomes yellow e ME buffer allows Na concentrations up to 20 mM which greatly favors reversible melting and still allows short run times for TGGE electrophoresis Mobile Cl ions which slow down the migration velocity of nucleic acids are avoided by using the sodium salt form of MOPS Due to their reduced mobility the large MOPS anions keep the current low TGGE System 29 Gel conditions 4 M urea can be used for low GC an
106. vs temperature for different values of the retardation length parameter This plot can be used for direct comparison with TGGE experiments superpositions of plots generated with or without mismatched positions given are useful as a hint whether specific mismatched duplexes could be detected among homoduplexed DNA in a mixture of sample and reference double strands having undergone a denaturation renaturation cycle using either perpendicular or parallel TGGE e A half denaturation temperature plot showing the half denaturation temperature for each base This plot can also be used to estimate the destabilizing effect of mismatches on the surrounding part of the sequence a temperature shift of the TGGE transition can be expected if the lowest melting part of the sequence is directly affected by the mismatch Calculations can be done for oligonucleotides gt 15 bases or long double strands gt 50 bases respectively In the case of oligonucleotide mode a length dependent correction for the strand dissociation process is applied the temperature range is adapted as well We do not have sufficient experimental results to stringently check for this mode to give valid results but for the length range of about 20 nucleotides there is at least experimental evidence Using oligo mode with far longer sequences gives misleading results Graphics output is possible in Postscript HPGL GIF and PBM format numeric results are available as well
107. which overlaps the range of effective separation Program the temperature of Tiow for L1 first thick lane on the gradient block close to T1 and program the temperature of Thigh 5 C for L6 last thick lane on the gradient block close to T2 for the parallel TGGE run fig 24b TGGE System 72 9 TGGE SSCP 9 1 Running an SSCP on the TGGE TGGE can be used in combination with SSCP single strand conformation polymorphism to improve often dramatically the frequency of detecting SSCP markers TGGE SSCP is non radioactive because it utilizes silver staining detection SSCP relies upon the separation of single stranded DNA or RNA which have formed hairpin secondary structures Different conformations exhibit different electrophoretic mobilities The conformation which a particular single stranded molecule adopts is sequence dependent and mutations are detected by their influence upon the secondary structure and hence the altered electrophoretic mobility Ti tu Conformation of Conformation of Separation in gel iduk wild type DNA base substituted electrophoresis Figure 25 Effect of conformational differences As indicated in figure 25 conformational differences between two different base substituted fragments can be achieved only within a limited temperature range Tiow T high At temperatures below Tiow both fragments adopt the characteristic hairpin structure On the other hand at temperatures higher than T high neithe
108. wo pre cut and pre soaked electrode wicks must be positioned at the start and the end of the polyacrylamide gel Wick and gel have to overlap see figure 16 Figure 16 Side view of the polyacrylamide gel on top of the gradient block during pre run Pay attention to the position of electrode wicks on top of the polyacrylamide gel Polybond film slot of polyacrylamide gel e Avoid any contact between sample slots and electrode wicks Otherwise the samples will diffuse into the electrode wicks e Load the samples quickly at room temperature without air bubbles Do not start the temperature gradient the temperature gradient is established after the samples have fully entered the polyacrylamide gel glass plate with 1 rectangular slot 50 ul volume approx 50 ng DNA RNA of interest 2 marker slots 5 ul volume 3 5 ng DNA RNA of interest If less volumes fill up the slots with running buffer or loading buffer to the volumes listed on top This will create better results TGGE System 37 VAN The time between mounting the gel onto the gradient block and loading the sample must not exceed 5 minutes e Close the safety lid of the electrophoresis chamber and start electrophoresis at 20 C or 25 C and 250 V for 2 5 min Standard electrophoresis conditions are given in chapters 5 3 and 6 1 e Make sure that the orientation of the gel and the safety lid is exact as indicated in the following
109. yl Glide e For parallel TGGE the removable electrophoresis buffer chambers must be positioned as indicated in figure 19 Platform surrounding the gradient block Electrophoresis buffer chamber with connectors for cathode Gradient block 4 Electrophoresis buffer chamber with connectors for anode Figure 19 Orientation of the electrophoresis chambers for parallel TGGE e Fill in the running buffer e g 0 1 x conc TBE into each electrophoresis chamber N Before you place the gel onto the gradient block be sure that the sample is ready for loading and cover film is available e The polyacrylamide gel attached to the Polybond film must be positioned as indicated in figure 20 The slots of the gel should be positioned at the beginning of the gradient block The first marked line L1 represents the beginning of the linear range of the gradient block TGGE System 40 BE ee an Soo ber SS Sa SE EC L ee Figure 20 Orientation of the polyacrylamide gel attached to the Polybond film on the gradient block during parallel TGGE See the position of the gel slots relative to the marked lines of the gradient block e Two pre cut and pre soaked electrode wicks must be positioned at the start and the end of the polyacrylamide gel Wick and gel have to overlap see figure 16 Figure 16 Side view of the polyacrylamide gel on top of the gradient block during pre run Pay

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