DYEnamic ET Primer DNA Sequencing Reagents for MegaBACE
Contents
1. 61 68 1996 Bashkin J et al Applied and Theoretical Electrophoresis 6 23 28 1996 Carrilho E et al Anal Chem 68 3305 3313 1996 Goetzinger W et al Electrophoresis 19 242 248 1998 Huang X C et al Anal Chem 64 967 972 1992 Huang X C et al Anal Chem 64 2149 2154 1992 Marsh M etal J Capillary Electrophoresis 4 83 89 1997 Mathies R A and Huang X C Nature 359 167 169 1992 u J etal Proc Nat Acad Sci USA 92 4347 4351 1995 abor S and Richardson C C Proc Nat Acad Sci USA 92 6339 6343 1995 abor S and Richardson C C Proc Nat Acad Sci USA 84 4767 4771 1987 abor S and Richardson C C J Biol Chem 264 6447 6458 1989 Sanger F et al Proc Nat Acad Sci USA 74 5463 5467 1977 Huibregtse J M and Engelke D R DNA and Protein Engineering Techniques 1 39 41 1988 McMahon G et al Proc Nat Acad Sci USA 84 4974 4978 1987 Carothers A M et al Biotechniques 5 494 496 498 499 1989 Murray V Nucleic Acids Research 17 8889 1989 Levedakou E N et al Biotechniques 5 438 442 1989 Lee J S DNA Cell Biol 1 67 73 1991 Messing J Methods in Enzymology 101 20 78 1983 Mead D A and Kemper B in Vectors A Survey of Molecular Cloning Vectors and Their Uses Butterworth Publishers Massachusetts USA 1986 Holmes D S and Quigley M Anal Biochem 114 193 197 1981 Dente2. T termination mix US79589 OR MegaBACE 7 deaza dGTP sequencing blend US79657 contains the following four termination mixes in separate vials A termination mix US79590 C termination mix US79591 G termination mix US79592 T termination mix US79593 Components of the kit continued The reagents comprising this product should be stored at 15 C to 30 C NOT Materials not supplied continued MegaBACE 1 000 sequencing instrument US79475 MegaBACE 40 ET Forward Primer Set GTT TTC CCA GTC ACG ACG US79880 MegaBACE 21 M13 ET Forward Primer Set TGT AAA ACG ACG GCC AGT US79970 MegaBACE 28 M13 Reverse 1 ET Primer Set AGG AAA CAG CT A TGA CCA T US79890 MegaBACE 28 M13 Reverse 2 ET Primer Set AGG AAA CAG CT A TGA CAT G US79900 MegaBACE 29 M13 Reverse ET Primer Set CA GGA AAC AGC TAT GAC C US79850 MegaBACE SP6 ET Forward Primer Set ATT TAG GTG ACA CTA TAG US79855 MegaBACE T7 ET Forward Primer Set TAA TAC GAC TCA CTA TAG GG 7 5 M ammonium acetate Ethanol 100 and 70 For sample preparation for injection p4 in a frost free freezer All reagents should be kept on ice when removed from storage for use For convenience the kit can be stored at 2 4 C for up to three months with no loss of performance however this should be avoided if the reagents will not be completely consumed within three months World wide web address http www amershambiosciences com Visit
3. tions PEETA DYEnamic ET Primer E79000M DNA Sequencing Reagents for MegaBACE Warning For research use only Not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Amersham 74000020 Ed AC 1999 01 e Biosciences Handling Storage Store at 15 C to 30 C p2 Page finder Components of the kit 3 Quality control cece tas eae eee oe 3 Materials not supplied 3 World wide web address 0 000000 4 Safety warnings and precautions 5 INTODUCHOMN y esis eia oe Ep eana ee k 6 Protocols Preliminary preparations and general handling instructions saaana aaa 10 1 Sequencing with DYEnamic ET Primer DNA Sequencing Reagents for MegaBACE 10 2 Preparation of master mixes 11 3 Preparation of termination reactions 11 4 Post reaction clean up Preparation of sequencing samples for capillary injection 12 5 Instrument setup and analysis of data 13 Appendices 1 Preparation of template DNA general considerations 14 2 Cycling parameters and quantity of template 15 3 Injection and run parameters 16 Troubleshooting oari ee an ee ae 19 References inate radiadi ee ls ee 26 LICENSES ected els et iae ea hE 27 Trademarks ss ieac cece eee 28 Quality control All batches of DYEnamic ET Primer DNA Se
4. 50 100 3 There was residual salt in the samples This can affect the ionic strength of the sample and inferfere with electrokinetic injection p19 Confirm that the recommended salt was used for ethanol precipita tion and that a wash step was performed If products were prepared using spin columns confirm that they were eluted in water Some preparations of size exclusion chromatography media are pre swollen in a salt containing buffer and must be washed several times with water to remove the salt In some cases it may be neces sary to wash the dry media several times to remove residual ions that may interfere with injection Alternatively sequencing reaction pellets may be resuspended in water rather than formamide See Appendix 3 for additional information concerning guidelines for electrokinetic injection 4 The template DNA contained excess EDTA Resuspend the template DNA in water or in dilute buffer containing lt 0 1 mM EDTA 5 The template DNA was of poor quality Contaminants carried over with template preparations can precipitate and interfere with the efficiency of electrokinetic injection There is a strong correla tion between the quality of template DNA and the success of sequencing experiments 6 Either the quantity of template DNA or the number of cycles used for amplification was insufficient Increase either the amount of DNA used in the reaction or the number of cycles 7 The annealing temperature was
5. DNA polymerase rapid ly denatures during extended periods at high temperatures Number of cycles and quantity of template The number of cycles required for optimal results depends on both the amount fmol and purity of template DNA used as well as on the sensi tivity of the fluorescent detection method Using the protocol described in this booklet the minimum quantities of highly purified DNA that p15 have been successfully sequenced are 25 fmol 50 ng of M13mp18 DNA and 50 fmol 100 ng of pUC18 DNA For routine sequencing at least 50 fmol 100 ng of M13 or 150 fmol 300 ng of plasmid DNA is sufficient When small quantities of tem plate DNA are used signal intensity is strongly influenced by the number of cycles If template quantities are near the limits stated above the number of cycles should be increased Usually 20 30 cycles are adequate for good signal generation As noted in the Troubleshooting section if too much DNA gt 5 pmol is used short read lengths are likely Appendix 3 Injection and run parameters The quality of an electrokinetic injection is affected by a number of factors including the amount of template DNA the presence of excess salts the consistency of the samples within the plate the loading solu tion the electrophoresis run temperature and the nature of the separa tion matrix being used Controlling these variables is the best way to optimize the results from a capillary electrophore
6. L et al Nucleic Acids Research 11 1645 1655 1983 Birnboim H C and Doly J Nucl Acids Res 7 1513 1523 1979 p26 All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request NOTICE TO PURCHASER LIMITED LICENSE This kit is sold pursuant to Authorization from PE Applied Biosystems under one or more of the following US Patents 4 849 513 5 015 733 5 118 800 5 118 802 5 151 507 5 171 534 5 332 666 5 242 796 5 306 618 5 366 860 4 855 225 and corresponding foreign patents and patent applications The purchase of this kit includes limited non transferable rights without the right to resell repackage or further sublicense under such patent rights to use this kit for DNA sequencing or fragment analysis solely when used in con junction with an automated instrument for DNA sequencing or fragment analysis which has been authorized for such use by PE Applied Biosystems or for manual sequencing Purchase of this product does not itself convey to the purchaser a complete license or right to perform automated DNA sequence and fragment analysis under the subject patents No other license is hereby granted for use of this kit in any other automated sequence analysis instrument The rights granted hereunder are solely for research and other uses that are not unlawful No other license
7. may contain acrylamide a neurotoxin and suspected carcinogen Please follow the manufacturer s M aterial Safety Data Sheet regarding safe handling and use of these materials Introduction DY Enamic ET Primer DNA Sequencing Reagents for M egaBACE are formulated for use with DY Enamic ET Primers and M egaBACE 1 000 a powerful capillary based sequencing system designed to provide high throughput sequencing laboratories with unprecedented read lengths high pass rates and industry leading data quality The reagents in the kit are formulated for use with sequencing mixes containing either dGTP or 7 deaza dGTP for resolution of band com pressions For sequencing a mixture comprised of Thermo Sequenase DNA polymerase Reaction buffer A C G or T termination mix tem plate DNA and dye labelled primer is thermally cycled The reaction products are then precipitated with salt and ethanol and resuspended in a formamide loading solution for separation and detection using M egaBACE 1 000 sequencing instrument This protocol provides the methods necessary for sequencing with DY Enamic ET Primers and DY Enamic ET Primer DNA Sequencing Reagents for M egaBACE Capillary array electrophoresis In capilllary array electrophoresis narrow bore capillaries filled with a separation matrix are used to resolve DNA sequencing fragments Under the force of an electric field the fragments migrate into electro kinetic injection and through electrophore
8. the Amersham Biosciences home page for regularly updated product information Materials not supplied continued e Electrophoresis matrix Long read Matrix US79676 for capillary electrophoresis This is linear polyacrylamide or LPA Equipment e Liquid handling supplies Vials pipettes centrifuge and vacuum centrifuge All sequenc ing reactions should be run in plastic microcen trifuge tubes typically 0 5 ml or 96 well plates suitable for thermal cycling e Instrument This kit is designed for optimal performance with MegaBACE 1 000 sequencing instrument e Thermal cycler For thermally cycled incuba tions between 50 C and 95 C 1 100 cycles Safety warnings and precautions Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals All chemicals should be considered as potentially hazardous Only persons trained in laboratory techniques and famil iar with the principles of good laboratory practice should handle this product Suitable protective clothing such as labora tory overalls safety glasses and gloves should be worn Care should be taken to avoid contact with the skin or eyes if contact should occur wash immediately with water see M aterial Safety Data Sheet for specific recommendations Warning This protocol requires the use of ethanol a flammable liquid Gel reagents
9. volumes of 100 ethanol to the pooled samples and mix well Place in a freezer for 15 min or overnight Centrifuge the tubes in a microcentrifuge 12 000 rpm for 30 min at either room temperature or 4 Centrifuge 96 well plates for at least 30 min at 2 500 x g Note If plates are centrifuged at a lower relative centrifugal force the time of centrifugation must be increased Samples can routinely be centrifuged for 30 min at 3 100 x g Remove the supernatant by aspiration Wash the pellet with 200 ul of 70 ethanol Vortex briefly and centrifuge as described above Remove the supernatant by aspiration and air dry the DNA pellets for 10 15 min Alternatively the samples can be dried in a vacuum centrifuge for 2 3 min Do NOT over dry the DNA pellets Dissolve each pellet in 10 ul of Loading solution and vortex vigorously for 10 20 s to ensure com plete resuspension Briefly centrifuge to collect the samples at the bottoms of the tubes wells and to remove any bubbles Note The DNA pellet MUST be completely dissolved at this step for optimal sequencing results If a fixed angle rotor was used for centrifugation the DNA pellet will be on the side of the microcen trifuge tube This material must be washed to the bottom of the tube to ensure that the entire reaction product is loaded onto the gel he following chart presents a brief summary of the steps used in preparing samples for capillary injection Pool termination reactions 40 y
10. 0 kV s For samples containing smaller amounts lt 50 ng of template DNA both signal intensity and base calling accuracy can be improved by resuspending sequencing products in water H owever for customers with variable amounts of DNA in their sequencing reactions resuspending samples in formamide loading solutions may allow the use of a broader range of injection conditions Injection volume According to the protocol outlined in this booklet the dried DNA pellet from a sequencing sample should be dissolved in 10 ul of formamide loading solution The minimum volume for sample resuspension and injection is 5 ul If volumes lt 5 ul are used contact of both the elec trode and the capillary with the sample is likely to be inconsistent Larger resuspension volumes 10 30 ul can improve the results observed with samples containing an excess amount of template DNA by reducing over injection of sample into the capillary p17 Run parameters Using standard run conditions of 100 min at 9 kV average read lengths gt 500 bases with 98 5 accuracy can be expected with control templates These conditions allow for an instrument turn around time of 2 h To take advantage of the superior resolving power of the LPA matrix and to achieve the longest read lengths electrophoresis should be carried out for 200 min at 6 kV Even with these recommendations the quality and quantity of the template remain the most important factors affect ing rea
11. DNA should be purified for use Cesium chloride gradi ents polyethylene glycol PEG precipitation adsorption to glass columns and other common DNA purification methods all produce p14 DNA suitable for use in this system Although there are many popular protocols for purifying plasmid DNA from 2 ml to 10 ml cultures we have had consistent success using Qiagen boiling 22 23 and alkaline 24 mini prep methods Appendix 2 Cycling parameters and quantity of template The temperatures used for cycling the sequencing reactions are based on characteristics of the sequencing primer whereas the number of cycles required will depend on the quantity and quality of the template DNA The following guidelines should assist in choosing cycling parameters Cycling temperatures For a typical sequencing primer of 15 25 bases and a G C content of 50 the melting temperature will generally be 50 65 C We suggest using a three temperature cycling program denaturation at 95 C for no more than 15 s annealing at 45 55 C for 5 15 s and extension at 70 72 C for 30 120 s The duration of neither the annealing nor denaturation steps appears to be critical and even brief pauses at these temperatures seem to be effective If in doubt a wide temperature range should be chosen with brief pauses at the temperature extremes Although some users include an initial denaturation step at 95 98 C this should be avoided since Thermo Sequenase
12. Filter 4 585DF20 Beam splitter A 540DRLP Beam splitter B 6 00DRLP p13 Appendix 1 Preparation of template DNA general considerations Preparation of highly pure DNA template is the single most important factor affecting the quality of sequencing data produced with M egaBACE 1 000 sequencing system and the success rate achieved Template of suitable quality can be prepared using a variety of proce dures and commercial products Preparation of single stranded plasmid DNA Several published methods are available for preparing single stranded DNA from clones in M 13 vectors and hybrid plasmid phage phagemid vectors 20 21 Preparation of double stranded PCR product templates PCR products are typically very good sequencing substrates for the M egaBACE system since their concentrations are relatively uniform and they are relatively free of inhibitory contaminants and large amounts of unsequenced template DNA eg vector sequence that can interfere with injection For removal of PCR primers and nucleotides samples can either be diluted or treated with exonuclease and shrimp alkaline phosphatase using the PCR Product Pre sequencing Reagent Pack US70995 Preparation of double stranded plasmid DNA Sequencing double stranded templates with DY Enamic ET Primer DNA Sequencing Reagents for M egaBACE requires no changes in the protocol Alkaline denaturation is not required for these templates For optimal results plasmid
13. after electrokinetic injection Failure to observe any signal above background in all four channels can result from several factors related to either the sequencing chemistry or the instrument For instrument problems consult the M egaBACE p21 User s M anual and call the M olecular Dynamics Field Support office at 1 800 743 7782 for further details Possible causes solutions 1 The buffer plate rather than the sample plate was used for injection Repeat the injection using the sample plate 2 One or more reaction components were omitted from the reaction mix Verify that all reaction components primer buffer nucleotides enzyme and template DNA have been added to all termination reactions Problem Late starts and broad poorly resolved peaks are prevalent in the sequences Late appearance of the primer peak and poorly resolved sequencing fragment peaks are most often due to capillary overloading as the result of too much template DNA entering during electrokinetic injection Template DNA molecules compete with sequencing products for injec tion and overabundance of these large DNA molecules can cause a reduced or sudden drop in the capillary current Overloaded samples frequently have low signals since the peaks are broad and diffuse and it is common to misdiagnose overloading as insufficient signal Under optimal conditions detection of all samples should begin within a few minutes of each other Possible causes solut
14. ction by the sequencing instrument Since ET primers all absorb the laser light efficiently their effective fluorescence intensity is 2 12 times greater than primers with single dye labels DY Enamic ET Primers developed for M egaBACE use 5 carboxy fluorescein FAM as the donor dye and rhodamine 110 R110 C 6 carboxyrhodamine R6G also known as REG A N N N N tetramethyl 5 carboxyrhodamine TM R also known as TAM RA G and 5 carboxy X rhodamine RO X T as the acceptor dyes Thermo Sequenase DNA polymerase Thermo Sequenase is an exonuclease free thermostable DNA poly merase specifically engineered by Amersham Biosciences for cycle sequencing The enzyme readily accepts dideoxynucleotide termi nators 10 and generates bands of uniform intensity much like T7 Sequenase DNA polymerase 11 12 These characteristics along with its thermostability make it ideal for use in cycle sequencing and greatly facilitate data interpretation and accurate base calling using automated sequencing software The formulation of the enzyme includes thermostable inorganic pyrophosphatase TAP cloned from Thermoplasma acidophilum TAP hydrolyzes the inorganic pyrophosphate product of nucleotide polymerization thus preventing the occurrence of pyrophosphorolysis and subsequent loss of peaks from sequence data Cycle sequencing In traditional dideoxynucleotide sequencing synthesis of a new strand of DNA is initiated at a specific pri
15. d length and success rate p18 Troubleshooting Prior to diagnosing problems associated with the sequencing reaction chemistry operation of the M egaBACE instrument should be verified for optimal performance by injecting a plate of MegaBACE M13 DNA Sequencing Standards US79678 and carrying out electrophoresis according to the accompanying protocol If the average overall read length of this standard plate is lt 500 bases 98 5 accuracy the instrument may require some routine maintenance such as capillary cleaning or focusing For further details please contact the M olecular Dynamics Field Support office at 1 800 743 7782 Problem Sequencing signals are weak Weak signal strength occurs when insufficient dye labelled DNA sequencing product is injected into a capillary Although it may be the result of unsuccessful sequencing it may also be due to inefficient injection of the reaction products It is important to remember that electrokinetic injection into capillaries is more efficient if the ionic strength of the loading solution is low Possible causes solutions 1 The ionic strength of the loading solution was too high Electrokinetic injection into capillaries is more efficient if the ionic strength of the loading solution is low 2 The duration of electrokinetic injection was too short Confirm that the recommended injection conditions were used Change the injec tion conditions by increasing the duration of injection
16. eneration of sequencing p24 products Increasing the number of cycles beyond this range may encourage the formation of cycling artifacts 3 The concentration of primer was too high Reduce the concentra tion and repeat the procedure 4 Self annealing of the primer occurred In some cases the only effective solution is to choose a different primer Problem Localized broad single color peaks are prevalent in the sequences The appearance of large broad single color peaks in dye primer sequencing products may result from hydrolysis of the dye primer espe cially when a long pre denature cycling step gt 95 C for gt 30 s is used The rationale for including a long pre denature step is to completely denature double stranded DNA molecules thus increasing the amount of template strand available for annealing and extension In practice however this affords little if any improvement for plasmid sequencing and in fact only serves to hydrolyze the dyes on the primer and dena ture the DNA polymerase Possible causes solutions 1 The dye primers were hydrolyzed during a pre denature step Do not denature at temperatures gt 95 C or for periods longer than 30 s If problems persist please contact Amersham Biosciences Technical Service for assistance p25 References 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Ce Oean an rw ne Bashkin J et al J Capillary Electrophoresis 3
17. incorrect for the primer being used Check the primer melting temperature and adjust the annealing temperature accordingly 8 Too little primer was used The total recommended amount of primer is 3 pmol 0 5 pmol each of the R6G and R110 labelled primers 1 pmol each of the TM R and RO X labelled primers p20 Using lt 1 pmol of total primer will generally result in reactions with weak signal strength 9 The volumes of the reagents used were incorrect Check for the cor rect volumes of the reagents to be added 10 Reagent integrity was compromised Repeat the reaction using a control DNA to verify reagent integrity Problem Signal color in one or more of the channels is weak or non existent Absence of signal can result from incorrect instrument setup failure to combine or mix reagents properly prior to performing the sequencing reactions or loss of sequencing products during ethanol precipitation Possible causes solutions 1 The beam splitters and bandpass filters were not installed in the correct orientation Check for correct installation 2 One or more reaction components were omitted from the reaction mix Verify that all reaction components primer buffer nucleotides enzyme and template DN A have been added to all termination reactions 3 DNA sequencing products were not pooled or were precipitated improperly Follow the protocol exactly as described Problem No signal above background is observed
18. ions 1 Too much template DNA was used during electrokinetic injection causing overloading of the capillary Repeat the sequencing reac tions using less template To determine the optimal amount of p22 template to use perform a titration of the template over a 50 fold range 0 2 0 5 1 2 5 and 11 ul for example 2 The injection conditions were not optimal Confirm that the recom mended injection conditions were used Repeat the injection of the samples for a shorter duration 25 50 shorter 3 The injection voltage was too high Reduce the voltage by 25 50 4 Too little loading solution was used Increase the volume of the resuspended samples by 100 200 and re inject using the original parameters 5 The electrokinetic injection and voltage parameters used were not optimal for injecting samples from water If injecting from water evaporate the samples to dryness and inject from loading solution Problem Peak spacing changes during the run giving rise to the accordion effect Occasionally the current in a capillary will suddenly drop or undergo rapid fluctuations resulting in dramatic changes in the peak spacing Possible causes solutions 1 Samples were near the limits of overloading Follow the suggestions for overloading described above 2 Bubbles formed in the LPA matrix either during injection or during electrophoresis Repeat the injection p23 Problem Extensions appear short with read le
19. is granted expressly impliedly or by estoppel Further information on purchasing licenses to perform DNA sequence and fragment analysis may be obtained by contacting the Director of Licensing at PE Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 AMERSHAM BlOsciences IS LICENSED AS A VENDOR FOR AUTHORIZED SEQUENCING AND FRAGMENT ANALYSIS INSTRUMENTS NOTICE TO PURCHASER ABOUT LIMITED LICENSE he purchase of this kit reagent includes a limited non exclusive sublicense under certain patents to use the kit reagent to perform one or more patented DNA sequencing methods in those patents solely for use with Thermo Sequenase DNA polymerase purchased from Amersham Biosciences for research activities No other license is granted expressly impliedly or by estoppel For information concerning availability of additional licenses to practice the patented methodologies contact Amersham Biosciences Inc Director Business Development 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA US Patent numbers 4 962 020 5 173 411 5 409 811 5 498 523 5 614 365 and 5 674 716 Patents pending t The Polymerase Chain Reaction PCR is covered by patents owned by Roche Molecular Systems and F Hoffmann La Roche Ltd A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as Amersham Biosciences and Affiliates when u
20. l Glycogen 10 g l 2 ul 7 5 M ammonium acetate 4 yl 100 ethanol 140 ul or 3 volumes Freezer 15 min to overnight Microcentrifuge 30 min Remove supernatant 70 ethanol wash 200 pl Microcentrifuge 15 30 min Remove supernatant Air dry or speed vac spin 5 min Loading solution resuspension 10 ul per reaction 5 Instrument setup and analysis of data 5 1 For instrument setup and data analysis please refer to the instrument documentation supplied with the MegaBACE 1 000 sequencing instrument 5 2 Verify that the MegaBACE 1 000 instrument has been equipped with the following standard filter set for blue dye primer sequencing Beam splitters A 540DRLP B 600DRLP Bandpass filters 555DF20 Channel 1 525DF15 Channel 2 620LP Channel 3 585DF20 Channel 4 5 3 Set the instrument for a dye primer run with the parameters suggested below For additional infor mation concerning injection and run parameters see Appendix 3 on page 16 Electrophoresis Parameters Sample injection voltage 3 kV Sample injection time 40 s Run voltage 9 kV Run time 100 min emperature 44 C Fill Flush Parameters High pressue time 200 s Relaxation time 20 min Pre run time 5 min Pre run voltage 9 kV Matrix flush time 1 16 s Matrix flush time 2 7 s Low pressure time 240 s Optics Parameters PMT 1 750 volts or as set PMT 2 750 volts or as set Laser mode Blue Filter 1 555DF20 Filter 2 525DF15 Filter 3 620LP
21. l ddC 1 0 ul ddG 1 0 ul ddT Super Q water 2 26 ul 2 26 ul 2 26 ul 2 26 ul Thermo Sequenase 32 U l 0 1 pl 0 1 ul 0 1 ul 0 1 pl Total volume 5 0 ul 5 0 ul 5 0 ul 5 0 pl Preparation of termination reactions Transfer 5 ul of each termination master mix prepared in step 2 1 to a clean tube well Add 5 pl 3 2 3 3 3 4 of DNA template in water to each tube well for a final termination reaction volume of 10 ul Mix thoroughly by gentle pipetting After preparing the termination reactions cap the tubes or seal the plates and place the reactions into the pre programmed thermal cycler Start the cycling program The cycle parameters shown below are general guidelines Specific cycling parameters used will depend on the amount and purity of the template DNA For addition al information concerning cycling parameters see Appendix 2 on page 15 95 155 SME 15s 70 1 min Repeat for 20 30 cycles After cycling is complete centrifuge the tubes plate briefly to collect the reaction mixtures at the bottoms of the tubes wells p11 O Post reaction clean up Preparation of sequencing samples for capillary injection 4 1 4 2 4 3 4 4 4 5 4 6 4 7 p12 Pool the four termination reactions into a single tube Optional step To improve the efficiency of ethanol precipitation a carrier such as glycogen 10 20 ug may be added to the pooled termination reactions Add 0 1 volume of 7 5 M ammonium acetate and 3
22. ming site by a DNA polymerase in the presence of deoxynucleotide triphosphates dN TPs Extension of the strand continues until a chain terminating dideoxynucleoside triphosphate ddN TP is incorporated 13 By controlling the relative concentrations of dNTPs and ddNTPs the majority of the DNA frag ments generated will terminate at lengths within the resolving capacity of the gel matrix used for separation When the fragments are labelled ps with dyes fluorescent sequencing instruments can derive sequence information from the fluorescence emitted by the fragments as they move through a particular area of the gel When thermostable DNA polymerases are used sequencing reactions can be cycled through alternating periods of thermal denaturation primer annealing and extension termination to increase the signal levels generated from template DNA 14 19 For each cycle the amount of sequencing product available for analysis will be equivalent to the amount of primed template with the theoretical yield roughly equal to the number of cycles in practice each cycle is not 100 efficient and the amplification will be somewhat lower By using a thermostable enzyme such as Thermo Sequenase DNA polymerase programmed cycling can be performed with no need for additional enzyme A cycling protocol is especially useful when the amount of template is limiting or the sensitivity of the detection system is low The use of DY Enamic ET Primers and The
23. ngths limited to lt 350 bases Possible causes solutions 1 Too much template DNA was used in the sequencing reaction In some cases the use of too much template especially PCR product DNA can exhaust the supply of ddN TPs in the reaction If this occurs the sequence will suddenly fade before reaching 350 bases in length This problem is especially prevalent if excess primer is also used Use lt 1 pmol of template DNA and 5 pmol of primer for each sequence By using less template the concentration of any potential contaminants is also reduced 2 The extension step was too short Increase the time of the extension step in the cycling program to 2 4 min Problem Localized broad four color peaks are common in the sequence The appearance of large broad four color peaks extending off scale may result from amplification of DNA that occurs during the cycling process This is similar or identical to the formation of primer dimer bands and other artifacts often seen in PCR but in this case products are made with labelled primers These artifacts are distinguishable from stops in the sequence primarily by their magnitude Possible causes solutions 1 The annealing temperature was too low Use more stringent anneal ing conditions when cycling Increasing the annealing temperature from 50 C to 55 57 C is often effective 2 Too many cycles were used Use fewer total cycles Generally 20 30 cycles are sufficient for effective g
24. quencing Reagents for MegaBACE are assayed according to the protocols described in this booklet Reactions are run on MegaBACE 1 000 sequencing instrument Specifications for release are based on assessment of sequence by length of read accuracy and signal quality with gt 500 bases of readable sequence achieved Materials not supplied Reagents e Water Use only deionized distilled water for the sequencing reactions Sequencing primers The following DY Enamic ET primers have been formulated specifically for use with Components of the kit Solutions included in DY Enamic ET Primer DNA Sequencing Reagents for M egaBACE have been carefully formulat ed for optimal sequencing results Each reagent has been tested extensively and its concentration determined to meet Amersham Biosciences standards It is strongly recommended that all reagents supplied in the kit be used as described in this protocol The following components are included in this product Thermo Sequenase 32 U ul MegaBACE formulation E79000M MegaBACE reaction buffer 260 mM Tris 32 5 mM MgCl pH 9 5 US79607 Loading solution 70 formamide 1 mM EDTA US79916 The following reagents should be obtained for use with this protocol MegaBACE dG sequencing blend US79655 contains the following four termination mixes in separate vials A termination mix US79586 C termination mix US79587 G termination mix US79588
25. rmo Sequenase DNA polymerase in conjunction with a cycle sequencing format yields the highest quality sequence data with minimal reagent template and labor requirements Protocols Preliminary preparations and general handling instructions A thermocycler should be pre programmed as described on page 11 For additional information concerning cycling parameters see Appendix 2 on page 15 The following reagents should be thawed on ice prior to use DYEnamic ET Primer DNA Sequencing Reagents for MegaB ACE enzyme buffer and loading solution e MegaBACE termination mixes either MegaBACE dG sequencing blend or MegaBACE 7 deaza dGTP sequencing blend DYEnamic ET Primer Set for MegaBACE Template DNA Whenever possible tubes should be kept capped and on ice to minimize evaporation of the small volumes of reagents used Reagents should be dispensed using dispos able tip micropipettes and care should be taken to avoid contamination of stock solutions Reaction mixtures must be thoroughly mixed after each addition typically by pumping the solution two or three times with a micropipettor without creating air bubbles Tubes plates should be centrifuged briefly to collect the reaction mixtures at the bottoms of the tubes wells A final brief centrifugation step should be performed to remove any bubbles prior to injection into the sequencing instrument 1 Sequencing with DYEnamic ET Primer DNA Sequencing Reagents for MegaBACE Thi
26. s protocol is a recommended starting point Further experiments to optimize the amount of primer and template DNA and conditions for thermal cycling may be required If changes in reaction volume are necessary the volume of reaction buffer used in the termination reactions should be adjusted Optimal results are obtained when the final MgCl concentration in each termination reaction is 2 4 mM 1 1 Dilute the DNA template in deionized water or in buffer containing no more than 0 1 mM EDTA The recommended quantities of template DNA are 100 500 ng 40 200 fmol of single stranded M13 DNA or 200 800 ng 80 400 fmol of double stranded DNA For additional information concerning preparation of DNA templates see Appendix 1 on page 14 p10 2 Preparation of master mixes 2 1 For each template to be sequenced prepare the following master mixes by combining an A C G or T termination mix from either MegaBACE dG sequencing blend or MegaBACE 7 deaza dGTP sequencing blend with the appropriate primer When all the reagents have been dispensed mix thoroughly by gentle pipetting The table below shows volumes for one sequencing reaction Adjust the volumes accordingly for additional reactions 3 1 Master Mixes A G T Reaction buffer 0 64 ul 0 64 ul 0 64 ul 0 64 ul ET Primer R6G 0 5 uM 1 0 pl ET Primer R110 0 5 pM 1 0 ul ET Primer TMR 1 0 pM 1 0 ul ET Primer ROX 1 0 uM 1 0 pl Termination mix 1 0 pl ddA 1 0 u
27. sed in conjunction with an authorized thermal cycler T7 Sequenase DNA polymerase This reagent kit is covered by or suitable for use under one or more US Patent numbers 4 795 699 4 946 786 4 942 130 4 962 020 4 994 372 5 145 776 5 173 411 5 266 466 5 409 811 5 498 523 and 5 639 608 Patents pending in US and other countries Exo and Shrimp Alkaline Phosphatase US Patent 5 756 285 Energy Transfer dyes and primers US Patent numbers 5 654 419 6 688 648 and 5 707 804 Thermo Sequenase DNA polymerase Patent pending Amersham Biosciences Inc 1999 All rights reserved Printed in the United States p27 Trademarks Amersham is a trademark of Nycomed Amersham plc DYEnamic MegaBACE Sequenase and Thermo Sequenase are trademarks of Amersham Biosciences Limited or its subsidiaries Qiagen is a trademark of Qiagen Inc Super Q is a trademark of Millipore Corporation p28 Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA tel 0870 6061921 fax 01494 544350 Amersham Biosciences AB SE 751 84 Uppsala Sweden tel 46 0 18165000 fax 46 0 1816 6458 Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA tel 1 800 526 3593 fax 1 800 329 3593 or 877 295 8102 Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiberg Germany tel0761 4903 01 fax0761 4903 405 Amersham e Biosciences
28. sis the capillaries and are separated according to size Because of their large surface to volume ratio capillaries allow efficient removal of heat generated during elec trophoresis Since higher voltages can be used fragments migrate more rapidly and separation times are shorter p6 The M egaBACE system uses six arrays of 16 capillaries each to provide rapid parallel separation of dye labelled DNA fragments from a total of 96 samples The capillaries are filled with a replaceable non crosslinked linear polyacrylamide LPA matrix By applying a variable time and variable voltage pulse the instrument can electrokinetically inject all 96 samples from a 96 well plate simultaneously Each capillary has a clear detection window located at a fixed distance from the sample loading point During electrophoresis the instrument uses laser excitation and a patented confocal optical detection system to both excite and detect the dye labelled DNA fragments as they migrate past the window 1 8 Energy transfer dye primers Energy transfer ET dye primers are uniquely designed oligonucleotides with two fluorescent dyes attached to each primer molecule for improved detection 9 The donor dye absorbs light of the wavelength of the argon ion laser in the sequencing instrument and transfers this absorbed light energy to the acceptor dye The acceptor dye then emits the absorbed energy as fluorescence at its characteristic emission wave length for dete
29. sis system Although general guidelines are provided below injection protocols should be optimized for each sample Injection solution voltage and duration The ionic strength of the solution used to dissolve DNA sequencing samples can dramatically affect the electrokinetic injection parameters A broad range of injection parameters can be used with samples dis solved in formamide loading solutions For these samples baseline injection conditions of 2 kV for 60 s are an appropriate starting point If signals are weak and read lengths are short the duration of injection can be increased If data lack resolution due to template overloading p16 the resuspension volume can be increased or the duration of injection can be reduced see Troubleshooting section The longest read lengths are obtained when the product of the time and the voltage of injection ranges from 50 to 200 kV s For the greatest reproducibility the applied injection voltage should not exceed 5 kV Sequencing samples may also be dissolved in water for injection If spin columns or gel filtration plates are used rather than salt ethanol precipi tation it is convenient to leave the samples in the eluent and directly inject from water H owever the range of voltage and duration of injec tion used with samples injected from water is typically narrower A good starting point for injection from water is 2 kV for 20 s with opti mal results typically obtained between 25 and 5
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