Animaltype Pig PCR Amplification Kit
Contents
1. 3 min denaturation at 95 C cooling at 4 C for analysis load 10 uL of the Matrix Standard into a 96 well reaction plate well A1 D1 Example for 16 Capillaries ABI 3130xl Composition Volume Hi Di Formamide 190 uL Matrix Standard 10 0 uL 3 min denaturation at 95 C cooling at 4 C for analysis load 10 uL of the Matrix Standard into 96 well reaction plate well A1 H1 and A2 H2 Performing Spectral Calibration Run nsert the 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection Software click New in the window Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol for Spectral Calibration Protocol Editor Name e g Spectral36_POP4_DS30 Type SPECTRAL Dye Set D Polymer POP4 Array Length 36 Chemistry Matrix Standard Run Module Spect36_POP4_1 Select OK to complete the Protocol Editor dialog box Animaltype Pig December 2007 In the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box Plate Editor for Spectral Calibration 1 New Plate Dialog Name e g Spectral DyeSet D Date Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name Select OK to complete the New Plate dialog box A new table in the Plate Editor opens automatically For further analyses for spectral calibration use with the same plate setting click Import select the xml file and click Open Plate Ed2. d n c 8 8 EE gz 8 2 3 8 8 3 3 3 S 3 ET a e e e ga e E S a ae a E x EJ a o a a a BE gii TE s a a e 8 8 g s a a 2a a a a e 115 t he e a a a a a D a D 5 ga e 5 x T i E m z c 118 Fi 2 3 pa 2 8 2 2 Elle i MT es T E eu El m m 912 2 s lg ae _ pen pes ole 5 E e f a a al i c E c ale a STE w w a a a a a a 2 8 8 2 p ha PS TN amp Quel a a eo x Fig 8 Electropherogram of the Animaltype Pig Allelic Ladder analysed on an ABI PRISM 310 Genetic Analyzer Allele assignment was performed using the Genotyper Software and the Animaltype Pig Template File Animaltype Pig December 2007 Table 5 Fragment Lengths of the Allelic Ladder Animaltype Pig analysed on an ABI PRISM 310 Genetic Analyzer Blue Panel Size bp Size bp Allele ABl 310 Further Alleles Allele ABI 310 Size bp ABI 310 Remarks Allele Further Alleles SBH2 6 FAM SBH4 6 FAM 50655 6 FAM 6 84 0 47 3 317 4 1 0 bp 5 416 6 21 139 2 49 1 322 7 1 0 bp 6 420 6 22 143 2 23 50 1 327 3 1 0 bp 7 424 7 24 151 0 51 3 332 6 x 1 0 bp 9 432 5 25 154 9 53 337 2 1 0 bp 0 436 8 26 1
3. 2 Electropherogram with Raw Data of the Matrix Standard 6 FAM Select analysis range with flat baseline Re inject the matrix sample if necessary Note start and end value Data Points of the analysis range e g start value 3400 end value 6400 Calculate the difference value e g 6400 3400 3000 Data Points Animaltype Pig December 2007 Generation of a New Matrix FILE NEW MATRIX as shown in Fig NE Make New Matrix Select the Matrix Standard Sample Files Number Of Dyes B 050128 FAM fsa Statat 050128 HEX fsa Statat v 050128 NED fsa Stat t 050128 ROX fsa Stat Points 300 Cancel Fig 3 Select Matrix Samples Import matrix samples for all dyes B G Y R Enter Start At value e g 3400 Enter difference value at Points e g 3000 Select OK to calculate the new matrix as shown in Fig 4 Matrix Biotype D mtx x Reactions B G af R EEN oons 0 0012 Josa72 10000 fosses 00107 ossoa fo ases 10000 0 048 o1273 o 1792 o 4864 10000 Fig 4 New Matrix Biotype 1 0000 m lt Save in the Matrix Folder FILE SAVE AS 0 Matrix Biotype Matrix Check Please check the new matrix with current samples FILE NEW PROJECT open run folder gt ADD SAMPLE FILES Select sample s in the column SAMPLE FILE SAMPLE INSTALL NEW MATRIX open ma
4. peaks for larger fragments Reduce the amount of template DNA to correct this imbalance Positive Control For the positive amplification control dilute the Control DNA DL 157 to 1 10 ng in the appropriate volume Instead of the template DNA pipette the diluted Control DNA into a reaction tube containing the PCR Master Mix Negative Control For the negative amplification control pipette Nuclease free Water instead of template DNA into a reaction tube containing the PCR Master Mix Animaltype Pig December 2007 1 2 Master Mix Preparation In order to activate the Multi Taq2 DNA Polymerase and to prevent the formation of non specific amplification products perform a hot start PCR reaction Standard Method recommended for all DNA samples Temperature Time 94 4 min hot start for Activation of the Multi Taq2 DNA Polymerase 94 205 60 40 5 30 Cycles 72 C 30 5 70 C 60 min 10 C hold Animaltype Pig December 2007 2 Electrophoresis using the ABI PRISM 377 DNA Sequencer For general instructions on instrument setup matrix generation and application of the GeneScan analysis Software please read the ABI PRISM 377 DNA Sequencer User s Manual Electrophoresis by using the GeneScan Software is described below For the combined application of the four fluorescent dyes 6 FAM HEX NED and ROX also called DS 30 the use of the virtual Filter Set D is allocated Generally the Filter Sets A and F are
5. yellow panel Allele Further Alleles Allele en Further Alleles SBH13 NED SBH22 NED 8 98 7 18 291 7 9 102 8 19 295 7 10 107 0 20 299 7 11 111 2 22 307 7 12 115 6 23 3117 13 1199 23 3 314 7 24 14 124 2 24 3 318 7 25 25 3 15 128 5 28 332 3 27 3 16 1327 17 136 9 SBH19 NED 18 1409 10 386 5 11 390 7 387A12F NED 2 394 7 9 205 4 3 399 0 10 1 210 3 11 14 403 0 124 218 3 5 407 0 13 2214 6 4110 13 1 2224 141 226 6 14 15 229 8 15 12 16 234 0 16 1 235 0 17 238 2 17 1 18 242 2 191 2472 20 250 2 21 254 2 a Biotype DNA pool b see Special Features chapter 5 Animaltye Pig December 2007 29 30 6 Interpretation of Results As mentioned above post PCR analysis and automatic allele allocation with suitable analysis software ensure a precise and reliable discrimination of alleles Pull up Peaks If peak heights are outside the linear detection range 23000 RFU or if an incorrect matrix has applied pull up peaks can occur at positions of a specific peaks in all colour channels In order to avoid pull up peaks peak heights should not exceed more than 3000 RFU Stutter Peaks Appearance of stutter peaks depends on the sequence of the repeat structure and on the number of alleles n 4 peaks are due to a loss of a repeat unit during amplification of tetranucleotide STR motives caused by slippage effects of the Multi 2 DNA Polymerase Interpretation of those peaks should be done in accordanc
6. 2007 3 Electrophoresis using the ABI PRISM 310 Genetic Analyzer For general instructions on instrument setup matrix generation and application of the GeneScan or GeneMapper ID Software please read the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis by using the GeneScan Software is described below For the combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 the use of the virtual Filter Set D is allocated Generally Filter Sets A and F are suitable too Material Capillary 47 cm 50 um green Polymer 310 Genetic Analyzer POP 4 Buffer 10x Genetic Analyzer Buffer with EDTA 3 1 Matrix Generation Prior to any analysis of DNA fragment size a matrix with the appropriate four fluorescent labels has to be generated Suitable matrix standards can be purchased from Applied Biosystems Dye Color Matrix Standard Order Number Blue B 6 FAM Applied Biosystems 401546 Green G HEX Applied Biosystems 401546 Yellow Y NED Applied Biosystems 402996 Red R ROX Applied Biosystems 401546 To generate useful matrix files it is necessary to perform four electrophoresis runs with the matrix samples PCR fragments labelled with 6 FAM HEX NED and ROX The runs have to be done under the same conditions like for the samples and the Allelic Ladders of the Biotype test kit Matrix sample Composition Volume Matrix sample 1 Hi Di Form
7. CL Let dea 8 2 Electrophoresis using the ABI PRISM 377 DNA GS CUEMGGlsssresase saves 9 2 1 Polyacrylamide Gel 595 esca cene det tede 9 2 2 Sample 9 2 3 Setting for GeneScan Software eee tnn 10 3 Electrophoresis using the ABI PRISM 310 Genetic Analyzer 11 3 1 Matrix 11 3 2 Sample adi uen ac ra ra RE M pda ds 14 3 3 Setting for GeneScan Software 14 3 4 Analysis Parameter Reese coit ceteates been oet ea ines res 15 4 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer 16 4 1 Spectral Calibration Matrix 16 4 2 Sample Po Cte AGO er to dA 19 4 3 Setting for GeneMapper ID Software eene 19 4 4 Analysis Parameter Analysis Method sene 21 S ANAIVSIS 22 COMMONS pes o Suan Ratan bp aa 24 5 2 Fragment Lengths and Alleles easi dede iar tette HOP 24 6 Interpretation of Results nose eri s a Fete Dd ed red dra c P Rc cd Ho dett 30 Animaltype Pig December 2007 Protocols for PCR Amplification El
8. four samples must be pipette for analysis on 4 capillary analysers If less then four samples are analysed fill up the empty positions on the plate with 12 uL Hi Di Formamide For reliable allelic assignment on 4 capillary analysers one Allelic Ladder per capillary should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Pay attention to keeping ambient conditions as recommended by the instrument manufacturer 4 3 Setting for GeneMapper ID Software Performing Run Insert the 96 well plate prepare on the autosampler tray In the Protocol Manager of the Data Collection Software click New in the window Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol Protocol Editor Name Run36_POP4_DyeSet D Type REGULAR Run Module HIDFragmentAnalysis36_POP4_1 Dye Set D for detailed description see Setting of the Run Module on the next page Select OK to complete the Protocol Editor dialog box Animaltype Pig December 2007 20 Previous to the first run it is necessary to edit the Run Module as follows In the Module Manager of the Data Collection Software click New to open the Run Module Editor dialog box Run Module 24min 50 500bp Run Modul Editor Value Oven Temperature C 60 Poly Fill Volume 4840 Current Stability uA 5 PreRun Voltage kV 15 PreRun Tim
9. 00 Default Sample or Allelic Ladder g SST550_50 500bp e g Animaltype Panels v1 e g Analysis HID 3130 select results group Run36_POP4_DyeSet D setting typed before For each of the columns click the column header to select entire column then select Edit Fill Down to apply the information to all of the selected samples and enter OK In Run Scheduler click Find All select link to link up the reaction plate on the autosampler with the newly created plate record position A or B and start the run During the run view Error Status in the Event Log or examine the quality of the raw data for each capillary in the Capillaries Viewer or the Cap Array Viewer View data as overview in Run History or Cap Array Viewer of the Data Collection Software Run data are saved in the Run Folder of the former chosen Result Group 4 4 Analysis Parameter Analysis Method The recommended settings in the worksheet Peak Detector are Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Pt 2000 Stop Pt 10000 Sizing All Sizes Smoothing Light Baseline Window 51 pts Local Southern Method Peak Amplitude Thresholds Bi wx G Min Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Slope Thresholds 0 0 The peak amplitude threshold Cutoff value corresponds to the minimum peak height that will be detected from the GeneMapper ID Software The thresholds are usually 50 200 RFU
10. 44 Renard C Vaiman M Chiannilkulchai N Cattolico L Robert C Chardon P 2001 Sequence of the pig major histocompatibility region containing the classical class genes mmunogenetics 53 490 500 Animaltye Pig December 2007 31 32 Notes Animaltye Pig December 2007
11. 58 9 54 341 5 x 1 0 bp 1 440 8 27 163 0 55 1 347 6 1 0 bp 2 445 2 28 167 1 56 349 1 x 1 0 bp 3 449 4 29 171 3 57 353 0 1 0 bp 4 453 8 30 175 5 58 357 0 1 0 bp 15 458 1 31 179 7 59 360 7 1 0 bp 6 462 2 32 183 9 60 364 6 1 0 bp 7 466 5 33 188 0 61 368 4 1 0 bp 8 470 8 34 192 2 35 62 372 2 x 1 0 bp 22 487 6 64 380 2 1 0 bp SBH18 6 FAM 65 1 385 8 1 0 bp 9 214 4 66 1 389 7 1 0 bp 11 222 5 12 226 6 13 230 8 14 235 0 15 239 2 16 243 5 17 247 3 18 251 3 19 255 2 20 259 2 21 263 2 22 267 1 23 271 0 Animaltye Pig December 2007 28 Table 6 Fragment Lengths of the Allelic Ladder Animaltype Pig analysed on an ABI PRISM 310 Genetic Analyzer Green Panel Allele 1 Further Alleles Allele Sze 100 Further Alleles SBH23 HEX SBH1 HEX Y 83 4 7 283 8 ge X 93 4 X 94 4 9 292 0 10 296 0 SBH20 HEX 11 300 0 19 136 0 12 304 2 20 139 9 13 308 4 21 143 6 14 312 6 2 0 bp 22 1473 15 316 8 2 0 bp 23 1514 16 320 9 24 1548 17 324 9 25 159 7 18 329 1 26 163 6 28 171 2 SBH10 HEX 29 1755 31 363 5 30 1795 32 367 5 31 183 5 33 371 5 32 187 5 34 375 7 33 191 6 35 379 9 34 195 6 36 383 9 35 199 4 37 388 0 36 203 3 38 392 0 39 37 207 3 38 40 400 0 39 214 8 41 404 0 40 218 6 42 408 0 41 2226 43 412 0 43 230 6 44 4158 45 49 254 1 46 423 8 47 427 8 48 431 8 49 50 439 8 Animaltype Pig December 2007 Table 7 Fragment lengths of the Allelic Ladder Animaltype Pig analysed on an ABI PRISM 310 Genetic Analyzer
12. 60 00 80 00 160 00 190 00 240 00 220 00 330 00 360 00 400 00 550 00 70 00 100 00 200 00 Fig 6 Electropherogram of the DNA Size Standard 550 ROX Lengths of Fragments in bp Allele designation of analyzed samples should be carried out either manually or with suitable analysis software e g GeneMapper ID or Genotyper Software in combination with the Animaltype Pig Template File from Biotype Template Files can be received as free downloads from our homepage www biotype de or as CD ROM on request Animaltype Pig December 2007 23 Special Features SBHA In rare cases deviation of 0 6 bp between signals versus the Allelic Ladder due to single nucleotide polymorphisms SNPs within repeating units can be observed Exact assignment of all alleles according to the Allelic Ladder is achieved by setting the tolerance to 1 0 bp in the analysis software This point is already fixed in Biotype Template Files for Genotyper or GeneMapper SBH1 Sometimes deviation of the signals for allele 14 and 15 versus the Allelic Ladder due to SNPs within repeating units can be observed Exact assignment of these alleles according to the Allelic Ladder is achieved by setting the tolerance to 2 0 bp in the analysis software This point is already fixed in Biotype Template Files
13. Animaltype PCR Ampitication Kit Features and Benefits Animaltype Pig is the sole commercially available test kit for fast and reliable genotyping of pigs It includes eleven tetranucleotide markers see table 1 For the most important breeding stocks German Large White LW German Landrace GL and Pi train Pi the combined Power of Exclusion CPE of these STR Systems is equivalent or even better then the 15 dinucleotide STRs recommended for Germany or Austria see table 2 Table 1 Animaltype Pig Tetranucleotide STR Markers Locus Chromosomal Mapping 387A12F 12p14 15 0655 7p11 SBH1 1p13 SBH2 3p16 17 SBH4 6035 5 10 9 11 13 SBH13 13946 47 SBH18 16023 SBH19 17912 14 SBH20 18q13 23 SBH22 Xp24 SBH23 X Y gonosomal Manuscripts for publication of Loci SBH1 23 in preparation for 387A12F see Kiuchi et al 2002 for 50655 see Renard et al 2001 Table 2 Combined Power of Exclusion of STR Markers German Large White LW German Landrace GL and Pi train Pi Multiplex CPE total cPE GL cPE LW cPE Pi 11 4 STRs Biotype 0 9992 0 9997 0 9987 0 9998 15 2 STRs 0 9965 0 9965 0 9988 0 9978 Nechtelberger et al 2001 Animaltype Pig offers basically and essentially technical advantages Genotyping of tetranucleotide marker with the Animaltype Pig PCR Amplification Kit guaranties definite peaks see Fig 1 Moreover the test kit includes primers for amplification of the gender specific marker Amel
14. Genetic Analyzers Getting Started Guide Electrophoresis by using the GeneMapper ID Software is described below The 4 Capillary System is named ABI 3130 before ABI 3100 Avant the 16 Capillary System is named ABI 3130xl before ABI 3100 For the combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 the utilization of the Dye Set D is allocated Material Capillary 3130 capillary array 36 cm Polymer 3130 POP 4 polymer Buffer 10x Genetic Analyzer Buffer with EDTA 4 1 Spectral Calibration Matrix Generation Prior to any analysis of DNA fragment size a spectral calibration with the four florescent labels 6 FAM HEX NED and ROX has to be generated for each analyzer The spectral calibration creates a matrix to correct the overlapping of fluorescence emission spectra of the dyes Performing a spectral calibration can be divided into the following tasks Choosing and setting up the Spectral Calibration Standards Loading the standards on the 96 well reaction plate per capillary one sample Creating the instrument protocol for performing spectral calibration Protocol Manager Define the plate assembly within the plate editor Plate Manager Performing a spectral calibration run and reviewing data Animaltype Pig December 2007 Setting up the Spectral Calibration Standards Example for 4 Capillaries ABI 3130 Composition Volume Hi Di Formamide 47 5 UL Matrix Standard 2 5 UL
15. amide 12 5 uL Matrix Standard 6 1 0 uL Matrix samole 2 Hi Di Formamide 12 5 uL RUM Matrix Standard HEX 1 0 uL Matix s itiple 3 Hi Di Formamide 12 5 uL p Matrix Standard NED 1 0 uL Hi Di Formamide 12 5 uL Matrix Standard ROX 1 0 uL 3 min denaturation at 95 C cooling at 4 C for analysis load the samples on the tray Create a Sample Sheet and enter sample designation Animaltype Pig December 2007 Injection List for Matrix Generation Injection list Module File GS STR POP 4 1 mL D Matrix File NONE Size Standard NONE Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 24 prepare Matrix Standards always without DNA Size Standard ROX Analysis of the Matrix Samples Open GeneScan or GeneMapper ID Software FILE NEW PROJECT open current run folder gt ADD SAMPLE FILES Click a single matrix sample in the column SAMPLE FILE Mark SAMPLE RAW DATA Review the matrix samples for a flat baseline beyond the primer peak There should be at least five peaks with peak heights about 400 4000 Y in every matrix sample optimal range 1000 3000 as shown in Fig 2 GeneScan 3 7 6 FAM fsa EJ File Edit Project Sample Settings View windows Help x a jo 500 1000 1500 2000 2500 3000 3500 4000 4500 6000 5500 000 3400 Data Points X 2700 1800 E s o i 0 Tale T 2404 virer gt I Fig
16. and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher then the background noise of the baseline Smoothing and Baselining Size Calling Method Peak Detection Sometimes point alleles i e alleles with at least 1 bp difference to the next integer allele like 387A12F allele 13 and 13 1 or 16 and 16 1 can not be distinguished For improved peak detection minimize the Peak Window Size further on Animaltype Pig December 2007 22 5 Analysis For general instructions on automatic sample analysing please read the GeneScan or GeneMapper ID Software User s Manual The determination of the exact lengths of the amplified products depends on the type of device on the conditions of electrophoresis as well as on the DNA Size Standard used Due to the complexity of some STR loci the size determination should be based on evenly distributed points of references Thus please use the DNA Size Standard 550 ROX with the following lengths of fragments 50 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp Tt 80 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 DNA Size Standard 550 fsa 17 Red 600 400 200 E 50 00 T 140 00 20 00 290 00 20000 24000 22500 28000 476 00 500 00 d
17. e s 180 Injection Voltage kV 3 0 Injection Time s 5 Voltage Number of Steps 40 Voltage Step Interval 15 Data Delay Time s 1 Run Voltage kV 15 0 Run Time s 1440 Apart from standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are to be recorded a shorter injection time may be selected For samples with low DNA content an injection time up to 20 s may be necessary Depending on the analysis conditions the Run Time for Animaltype Pig was modified in order to analyze lengths of fragments up to 500 bp Click Save As and enter the name of the new module g 24min 50 500bp and enter OK Click Close to exit the Module Editor Previous to every run it is necessary to compile the plate as follows n the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box GeneMapper Plate Editor I New Plate Dialog Name e g Plate DyeSet D Date Application select GeneMapper Application Plate Type 96 Well Owner Name Operator Name Select OK to complete the New Plate dialog box A new table in the Plate Editor opens automatically Animaltype Pig December 2007 21 GeneMapper Plate Editor 11 Column Name Sample Name Priority Sample Type Size Standard Panel Analysis Method Snp Set User defined 1 3 Results Group 1 Instrument Protocol 1 Type name for the samples e g 1
18. e for Animaltype Pig was modified in order to analyze lengths of fragments up to 500 bp Animaltype Pig December 2007 3 4 Analysis Parameter The recommended analysis parameters are Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds B Y R Min Peak Half Width 2 pts Polynorminal Degree 3 Peak Window Size 11 pts Size Call Range Min 50 Max 550 Size Calling Method Local Southern Method Split Peak Correction None The peak amplitude threshold Cutoff value corresponds to the minimum peak height that will be detected from the GeneScan or GeneMapper ID Software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher then the background noise of the baseline Sometimes point alleles i e alleles with at least 1 bp difference to the next integer allele like 387A12F allele 13 and 13 1 or 16 and 16 1 can not be distinguished For improved peak detection minimize the Peak Window Size further on Animaltype Pig December 2007 4 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the ABI PRISM Data Collection Software and the GeneMapper ID Software refer to the ABI PRISM 3130 3130xI
19. e with the Template Files of the Genotyper and GeneMapper ID Software Template independent Nucleotide Addition The terminal transferase activity of Multi Taq2 DNA Polymerase leads to the addition of an adenosine residue preferentially at the 3 end of amplified DNA fragments Incomplete extension is responsible for double peaks resulting from the absence of the terminal adenosine residue The artefact peak is one base shorter than expected 1 peaks All Biotype primers are designed to minimize these artefacts In addition the final extension step of 70 C for 60 minutes is included to the PCR protocol in order to reduce formation of artefacts Peak height of the artefact correlates with the amount of DNA Every laboratory should determine their own thresholds for analysis of the peaks Animaltye Pig December 2007 References B r W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W Olaisen B 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems nt J Legal Med 110 175 176 Kiuchi S Inage Y Hiraiwa H Uenishi H Yasue H 2002 Assignment of 280 swine genomic inserts including 31 microsatellites from BAC clones to the swine RH map IMpRH map Mamm Genome 13 80 88 Nechtelberger D Kaltwasser C Stur I Meyer JN Brem G Mueller M Mueller S 2001 DNA microsatellite analysis for parentage control in Austrian pigs Anim Biotechnol 12 141 1
20. ectrophoresis and Analysis 1 PCR Amplification 1 1 Master Mix Preparation The table below shows the volumes of all PCR reagents per 25 uL reaction volume including a DNA sample volume of 3 0 uL template DNA Determine the number of reactions to be set up positive control and negative control reactions should be included Add one or two reactions to this number to compensate the pipetting error Number of PCR Reactions Volume in UL 1 10 25 100 Nuclease free Water 14 1 141 0 352 5 1410 0 Reaction D 5 0 50 0 125 0 500 0 Primer Mix 2 5 25 0 62 5 250 0 Multi 2 DNA Polymerase hot start 2 5 U uL 0 4 4 0 10 0 40 0 Volume of Master Mix 22 0 220 0 550 0 2200 0 contains dNTP Mix BSA All components should be mixed vortex and centrifuged for about 10 s before preparation of the Master Mix The DNA volume applied to the assay depends on its concentration An increase of DNA volume to more than 5 uL is not recommended because potential PCR inhibitors may interfere Adjust the final reaction volume with Nuclease free Water to 25 uL Generally store DNA templates in Nuclease free Water or in diluted TE buffer 10 mM Tris HCI pH 8 0 and 1 mM EDTA e g 0 1x TE buffer The Primer Mixes are adjusted for balanced peak heights with 30 PCR cycles and 1 10 ng Control DNA DL157 in a reaction volume of 25 uL If more DNA template is introduced higher peaks will be expected for small PCR fragments and relatively low
21. for Genotyper or GeneMapper SBH23 Amelogenin Rarly deviation of 1 0 bp for the X specific signal versus the Allelic Ladder due to an insertion of a base can be observed Exact assignment of the X specific allele according to the Allelic Ladder is achieved by setting the tolerance to 2 0 bp in the analysis software This point is already fixed in Biotype Template Files for Genotyper or GeneMapper Unspecific amplificates Two artefact signals were observed in 6 of the DNA samples from Biotype Diagnostic GmbH at position 294 bp in 6 FAM between the allelic ranges of SBH18 and SBH4 and at position 298 bp in HEX within the allelic region of SBH1 These artefacts do not match with signals of the Allelic Ladder or with known alleles from the population study Animaltype Pig December 2007 24 5 1 Controls The Control DNA DL157 of the PCR Amplification Kit represents the following alleles Table 4 Allele Determinations of Animaltype Pig Locus Control DNA DL157 387A12F 13 21 50655 9 12 SBH1 14 16 SBH2 22 33 SBHA 56 62 SBH10 48 49 SBH13 13 15 SBH18 12 14 SBH19 14 14 SBH20 32 39 SBH22 20 23 SBH23 Y X 5 2 Fragment Lengths and Alleles Table 5 to table 7 display the values for fragment lengths of individual alleles that refer to the DNA Size Standard 550 ROX All analyses have been performed on an ABI PRISM 310 Genetic Analyzer with POP 4 polymer Different analysis instruments DNA Size Standards or poly
22. itor for Spectral Calibration 11 Column Sample Name Type name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_DS30 setting described before For each of the columns click the column header to select entire column then select Edit Fill Down to apply the information to all of the selected samples and enter OK n Run Scheduler click Find All select link to link up the reaction plate on the autosampler with the newly created plate record position A or B and start the run View Pass Fail Status after the run in the Event Log and open the Spectral Viewer to review and evaluate the spectral calibration profile for each capillary Info Raw Data EPT Data Matrix Standard DS 30 Biotype fsa 0 1000 2000 3000 4000 5000 6000 3000 80004 70004 60004 50004 Fig 5 Electropherogram with Raw Data of the Matrix Standard for DS 30 Note If all capillaries passed the test activate this Spectral Calibration file for Dye Set D in the Spectral Viewer Animaltype Pig December 2007 4 2 Sample Preparation Composition Volume Hi Di Formamide 12 3 uL DNA Size Standard 550 ROX 0 2 uL prepare 12 uL of the mix Formamide DNA Size Standard for all samples add 1 uL PCR product diluted if necessary or Allelic Ladder 3 min denaturation at 95 C cooling at 4 C for analysis load the samples on the tray Because injections proceed simultaneously for all capillaries
23. mers might result in different lengths of fragment These Data could serve as approximate allele sizes for the creation of a new analysis template In order to balance instrument specific deviations further fine tuning of the equipment should be done by measuring sample fragments of known lengths In addition a visual alignment with the Allelic Ladder is recommended Scaling Horizontal 75 505 bp Vertical Depending on signal intensity Animaltype Pig December 2007 25 Figure 7 009 0001 0081 099 pJepuejs YNG 005 Os 0 08 0009 nac 5 oze 00 087 097 Oz zz 00022 00 092 00 pz 00 00 00 Ore 00 00 00 092 00 ozz 00 06 ooz 081 00 081 00 001 00 1 0006 00 091 00 071 00 08 Pay 9 85426170 Big ed yeumx MOBA 9 ESPLO Big ed yeunx bd D usa 19 85326170 Big ed yeunx 5 zz ania 19 85329110 Big adAyeunuy 091 Opl 01 001 08 Fig 7 Electropherogram of the Animaltype Pig using 2 ng Control 0157 Analysis was done on ABI PRISM 310 Genetic Analyzer with the DNA Size Standard 550 ROX Allele assignment was performed using the Genotyper Software and the Animaltype Pig Template File September 2006 Animaltype Pig 26 Figure 8 2 2 g 2 e ae E EE 8 3 a a Tu a F Te F gl E
24. ndards DS 30 for ABI PRISM 310 Genetic Analyzer and Applied Biosystems 401546 and 402996 NED ABI PRISM 377 DNA Sequencer Matrix Standards DS 30 for ABI PRISM 3100 3130 Applied Biosystems 4345827 and ABI PRISM 3700 3730 Qiagen DNeasy Blood amp Tissue Kit 50 Preparations Qiagen 69504 Trademarks and Patents ABI PRISM GeneScan Genotyper GeneMapper and Applied Biosystems are registered trademarks of Applied Biosystems Inc 6 FAM HEX NED ROX POP 4 and Hi Di are trademarks of Applied Biosystems Inc GeneAmp is a registered trademark of Roche Molecular Systems The PCR is under patent law Patentees are Hoffmann La Roche Inc and F Hoffmann La Roche Roche DyeEx and DNeasy are registered trademarks of Qiagen Warning and Safety Instructions The PCR Amplification Kit contains the following potentially hazardous chemical Kit Component Chemical Danger Primer Mix Reaction Mix sodium azide NaN toxic if swallowed develops toxic gases and Allelic Ladder when it gets in contact with acids For the Material Safety Data Sheet MSDS of all Biotype products please contact us For MSDS of additional reagents to be needed please contact the corresponding manufactures Animaltype Pig December 2007 Content TEP CR AU UMC AUG coaster Raoul tando dut a e v de etd 7 Tal M ster ICE CG focus oct 7 1 2 Master Mix Preparation
25. ogenin SBH23 Finally Animaltype Pig is provided with an Allelic Ladder that allows standardisation of different laboratories C 236 241 3000 2000 1000 TTE TITTTT TYIYIT 220 240 220 240 220 240 fragment size bp Fig 1 The electropherogram displays the advantages of tetranucleotide STRs in comparison to dinucleotide STRs A 0005 Dinucleotide STR B SBH13 Tetranucleotide STR C SBH7 Tetranucleotide STR with 2 bp insertion not included in the test kit RFU relative fluorescence units Product Description Animaltype Pig PCR Amplification Kit is a multiplex application for kinship testing and determination of the gender In one PCR reaction the eleven polymorphic tetranucleotide Short Tandem Repeats loci 387A12F 50655 SBH1 SBH2 5 SBH10 SBH13 SBH18 SBH19 SBH20 and SBH22 as well as the gender specific marker SBH23 are amplified simultaneously Preferentially the test kit is employed for fast and reliable DNA genotyping of blood or tissue samples especially ear cartilage One primer for each locus is fluorescence labelled with 6 FAM SBH2 SBH18 SBH4 50655 HEX SBH23 SBH20 SBH1 SBH10 or NED SBH13 387A12F SBH22 SBH19 whereas a well balanced intensity of all signals was elaborated for the primer mix The detection limit of Animaltype Pig PCR Amplification Kit is less about 1 ng genomic DNA The use of 1 10 ng DNA is recommended Validation and evaluation of the test kit ha
26. rs Bar et al 1997 Animaltype Pig December 2007 Content Animaltype Pig PCR Amplification Kit 100 Reactions Nuclease free Water 3 0 mL Reaction Mix D 500 uL Primer Mix 250 uL DNA polymerase 40 uL Control DNA DL157 10 uL DNA Size Standard 550 25 uL Allelic Ladder 10 pL Ordering Information Animaltype Pig 25 Reactions Cat No 11 12110 0025 Animaltype Pig 100 Reactions Cat No 11 12110 0100 Animaltype Pig 400 Reactions Cat No 11 12110 0400 Animaltype Pig 1000 Reactions Cat No 11 12110 1000 Storage Store all components at 20 C and avoid repeated thawing and freezing Primer Mix and Allelic Ladder must be stored protected from light The DNA samples and post PCR reagents Allelic Ladder and DNA Size Standard should be stored separately from the PCR reagents The expiry date is indicated on the kit cover Quality Assurance The content of Biotype test kits is subjected to an intensive quality assurance of the Biotype Diagnostic GmbH The quality of test kits is controlled continuously in order to document the unrestricted usability For questions regarding the quality assurance please feel free to contact us Animaltype Pig December 2007 Additional Required Reagents In order to use the Biotype PCR Amplification Kit additional reagents are needed We strongly recommend the application of the following products Reagent Supplier Order Number Hi Di Formamide 25 mL Applied Biosystems 4311320 Matrix Sta
27. suitable too Prior to any analysis of DNA fragment size a matrix with the appropriate four fluorescent dyes has to be generated for the instrument Appropriate matrix standards can be purchased from Applied Biosystems 2 1 Polyacrylamide Gel 5 Composition Urea 30 Acrylamide bisacrylamide solution 29 1 10x TBE buffer Water Filtrate and degas solution 10 Ammonium persulfate TEMED Use glass plates with a spacing of 36 cm 2 2 Sample Preparation Composition Hi Di Formamide Blue Dextran DNA Size Standard 550 ROX PCR product diluted if necessary or Allelic Ladder 3 min denaturation at 95 C cooling at 4 C apply 1 5 uL sample to the gel Animaltype Pig Amount Volume 21 0g 8 4 mL 6 0 mL 20 0 mL 350 uL 15 uL Volume 1 8 uL 0 2 uL 1 0 uL December 2007 2 3 Setting for GeneScan Software Plate Check Module D PreRun Module GS PR 36D 1200 Run Module GS Run 36D 1200 Matrix D 6 FAM HEX NED ROX Standard SST550 ROX Programming of the Run Module for Biotype Test Kits 6 FAM HEX NED ROX Open program 377 Collection Open File New GeneScan Run Select the module GS Run 36D 1200 in the run window Click at the sheet symbol in the run window Use the following settings Parameter Setting Voltage 3000 V Current 50 0 mA tae anas Rd Save the module in the field Save Copy Gel temperature 51 C Laser power 40 0 mW Animaltype Pig December
28. trix folder and select new matrix Re Analyse your samples With the new matrix there should be no pull up peaks between the dye panels B G Y R Animaltype Pig December 2007 3 2 Sample Preparation Composition Volume Hi Di Formamide 12 3 uL DNA Size Standard 550 ROX 0 2 uL prepare 12 uL of the mix Formamide DNA Size Standard for all samples add 1 uL PCR product diluted if necessary or Allelic Ladder 8 min denaturation at 95 C cooling at 4 C for analysis load the samples on the tray Signal Intensities In order to increase the intensity of signals Reduce the volume of the DNA Size Standard 550 ROX the peaks of the Size Standard should be about 500 relative fluorescent units RFU Purify the PCR products with DyeEx 2 0 Spin Kit Qiagen 63204 3 3 Setting for GeneScan Software Create a Sample Sheet and enter sample designation Injection List Module File GS STR POP 4 1 mL D Matrix File z B Matrix Biotype Size Standard z B SST550_50 500bp Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 28 Apart from standard setting the injection time may be between 1 and 10 s depending on the type of sample If blood samples with very high signal intensities are to be recorded a shorter injection time may be selected For samples with low DNA content an injection time up to 10 s may be necessary Depending on the analysis conditions the Run Tim
29. ve been performed for the GeneAmp 9700 thermal cycler ABI PRISM 310 Genetic Analyzer and ABI PRISM 3100 3130 Genetic Analyzer Animaltype Pig December 2007 Application Proof of origin according to the EU Directive Kinship testing in context with control of breeding Status of inbreeding for herd book populations Table 3 Locus specific Information of Animaltype Pig Locus 38 A12F 50655 SBH1 SBH2 SBH4 SBH10 SBH13 SBH18 SBH19 SBH20 SBH22 SBH23 Y SBH23 X GenBank Accession AB059041 AJ251829 submitted submitted submitted submitted submitted submitted submitted submitted submitted submitted submitted Repeat Motif of the Reference Allele TTCT 2 CT TTCT o GGAA CTTT is AGAA 24 AA AGAA GAAA GGAA GAAA GAAA GAAG AAAG AGAG s AAAG s AA AAAG A AAAG AAAG A AAAG AG AAAG AGAG AAAG TAGA 5 TAGA TAGA TAGA CAAA TATC is 5 GTCT ATCT o CTTT 4 CTTT CTTC CTTT 2 CTTT a ATAG s ATG ATAG ATG ATAG Reference 48 15 15 24 20 Allele Range 9 21 5 22 7 18 6 34 47 3 66 1 31 50 8 18 9 23 10 16 19 49 18 28 The repeat motifs shown in Table 3 are concordant with the International Society for Forensic Genetics ISFG guidelines for the use of microsatellite marke
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