PCR Methods of Genotyping

Thomas J. Hudson1, Chris D. Clark2, Michele Gschwend2, Erica Justice‐Higgins3

1 Whitehead Institute, Cambridge, Massachusetts, 2 Stanford University School of Medicine, Stanford, California, 3 Massachusetts General Hospital, Charlestown, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.5
DOI:  10.1002/0471142905.hg0205s12
Online Posting Date:  May, 2001
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Abstract

Two protocols discuss labeling PCR products with radioactive labels. The PCR products are then analyzed on a denaturing polyacrylamide gel and visualized by direct autoradiography. The resulting band pattern is used to define the SSLP genotype of the individual. Another method of genotyping uses a simple silver staining technique to detect PCR‐amplified SSLPs electrophoresed in denaturing polyacrylamide gels. In a fourth protocol, multiple unlabeled PCR products from one individual are pooled, separated on a denaturing polyacrylamide gel, transferred to a nylon membrane, and sequentially hybridized to nonradioactive end‐labeled probes derived from the primers used to amplify each SSLP marker. The probes are detected on film using chemiluminescence. Support protocols describe the preparation of an M13 sequence ladder size standard and digoxigenin labeling of the probe, both of which are required for the chemiluminescent method.

     
 
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Table of Contents

  • Basic Protocol 1: PCR Amplification of SSLPs Using End‐Labeled Primers
  • Alternate Protocol 1: PCR Amplification of SSLPs Using [α‐32P]dCTP Internal Labeling
  • Basic Protocol 2: Nonradioactive Multiplex Analysis of SSLPs
  • Support Protocol 1: Preparation of M13 Sequence Ladder Size Standard
  • Support Protocol 2: Digoxigenin Labeling of Probes Using Terminal Transferase
  • Basic Protocol 3: Nonradioactive Analysis of SSLPs Using Silver Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR Amplification of SSLPs Using End‐Labeled Primers

  Materials
  • 20 µM forward and reverse primers (store at −20°C)
  • 10× T4 polynucleotide kinase buffer ( appendix 3E; omit BSA or gelatin and include 1 mM spermidine and 1 mM EDTA)
  • 10 mCi/ml [γ‐32P]ATP (3000 Ci/mmol)
  • 50 U/µl T4 polynucleotide kinase
  • Template DNA: 5 to 20 ng/µl genomic DNA in TE buffer or H 2O
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 5 U/µl Taq DNA polymerase
  • Light mineral oil
  • 2× formamide loading buffer ( appendix 2D; store in 10‐ml aliquots ≤6 months at −20°C)
  • 65°C water bath
  • 96‐well microtiter plate suitable for use in a thermal cycler
  • Centrifuge and rotor with microplate carrier (e.g., Beckman or Sorvall)
  • Thermal cycler accommodating 96‐well microtiter plate
  • Used X‐ray film or Whatman 3MM filter paper
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3F) and labeling of DNA ( appendix 3E)

Alternate Protocol 1: PCR Amplification of SSLPs Using [α‐32P]dCTP Internal Labeling

  • DNA template: 5 to 20 ng/µl genomic DNA in 10 mM Tris⋅Cl/0.2 mM EDTA, pH 7.3 (store at 4°C) recipe10× cold nucleotide mix (see recipe)
  • Primer mix: 100 ng/µl each forward and reverse primers (store at −80°C)
  • 10 µCi/µl [α‐32P]dCTP (800 Ci/mmol)

Basic Protocol 2: Nonradioactive Multiplex Analysis of SSLPs

  Materials
  • Template DNA: 10 ng/µl genomic DNA in H 2O from each individual to be genotyped
  • recipePrimer mix (see recipe) for each SSLP to be amplified
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • 2.5 mM 4dNTP mix ( appendix 2D)
  • 5 U/µl Taq DNA polymerase
  • Qiaex Gel Extraction Kit (Qiagen) containing solutions QX1 and QX3 and Qiaex glass bead suspension
  • 2× formamide loading buffer ( appendix 2D)
  • M13 sequence ladder size standard (see protocol 4)
  • TBE buffer ( appendix 2D)
  • recipePrehybridization solution (see recipe)
  • Digoxigenin‐labeled probes (made from primers used to amplify SSLPs and M13 anti‐universal primer; see protocol 5)
  • recipeOligo wash buffer (see recipe)
  • recipeBlocking solution (see recipe)
  • Alkaline phosphatase–conjugated anti‐digoxigenin Fab fragments (Boehringer‐Mannheim)
  • recipeDetection buffer (see recipe)
  • recipeSubstrate buffer (see recipe)
  • recipe0.2 mM CSPD (see recipe)
  • 2 mM EDTA/0.2% (w/v) SDS, preheated to 70°C (made from sterile stock solutions ( appendix 2D); store at room temperature)
  • 96‐well microtiter plates
  • 0.2‐ml thin‐walled PCR tubes arrayed in 96‐well (8 × 12) format
  • MicroAmp Full Plate Cover (Perkin‐Elmer)
  • Thermal cycler with heated cover, accommodating 0.2‐ml thin‐walled PCR tubes arrayed in 96‐well format, preheated to 94°C
  • 42°C water bath
  • Blotting filter paper (Owl Scientific Plastics)
  • TE 90 GeneSweep Sequencing Gel Transfer Unit (Hoefer)
  • Hybond‐N membrane (Amersham)
  • 80°C oven
  • Stratalinker (Stratagene)
  • 38 × 300–mm glass tubes, silanized (e.g., CPMB APPENDIX 3)
  • Hybridization oven (e.g., Model 400 Hybridization Incubator, Robbins Scientific)
Rolling shaker (e.g., Model V5250 Incubator, Robbins Scientific, or Model 400 set at room temperature)
  • Autoradiography film (e.g., Hyperfilm‐MP, Amersham)
  • 70°C incubator
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3F)

Support Protocol 1: Preparation of M13 Sequence Ladder Size Standard

  Materials
  • M13mp18 single‐stranded DNA in TE buffer ( appendix 2D)
  • 0.5 pmol/µl M13 universal sequencing primer (5′‐GTAAAACGACGGCCAGT‐3′; e.g., U.S. Biochemical)
  • recipe5× Sequenase reaction buffer (see recipe or purchase from U.S. Biochemical)
  • TE buffer ( appendix 2D)
  • 0.1 M DTT (dithiothreitol)
  • 7.5 µM 4dNTP mix ( appendix 2D)
  • 13 U/µl Sequenase Version 2.0 T7 DNA polymerase (U.S. Biochemical)
  • Termination mix: 80 µM each dGTP, dATP, dCTP, dTTP, plus 8 µM ddATP in 50 mM NaCl (prepared mix available from U.S. Biochemical)
  • 2× formamide loading buffer ( appendix 2D)
NOTE: M13 DNA, DTT, Sequenase reaction buffer, Sequenase T7 DNA polymerase, and termination mix are included in the Sequenase Version 2.0 DNA Sequencing Kit (U.S. Biochemical).

Support Protocol 2: Digoxigenin Labeling of Probes Using Terminal Transferase

  Materials
  • recipe5× terminal transferase reaction buffer, pH 6.6 (see recipe)
  • 25 mM CoCl 2
  • 6.6 µM forward or reverse primer used to amplify each SSLP (see protocol 3)
  • 6.6 µM M13 anti‐universal primer (5′‐ACTGGCCGTCGTTTTAC‐3′)
  • 1 mM digoxigenin‐11‐dUTP (digoxigenin‐11‐2′,3′‐deoxyuridine 5′‐triphosphate; Boehringer Mannheim)
  • 10 mM dATP
  • 50 U/µl terminal transferase (Boehringer Mannheim)
  • 0.2 M EDTA, pH 8.0 ( appendix 2D)
CAUTION: The potassium cacodylate in terminal transferase buffer is hazardous; see appendix 2A for safety guidelines.

Basic Protocol 3: Nonradioactive Analysis of SSLPs Using Silver Staining

  Materials
  • Denaturing polyacrylamide gel of PCR‐amplified SSLPs (see protocol 1, steps to )
  • 10% ethanol
  • 1% (v/v) nitric acid
  • recipeSilver nitrate staining solution (see recipe)
  • recipeDeveloping solution (see recipe)
  • 10% (v/v) acetic acid
  • Staining apparatus, e.g., see Figure
  • Whatman filter paper
NOTE: Use sterile, deionized, double‐distilled water in all recipes and steps. Use ∼200 ml of each solution in the following steps.
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Figures

Videos

Literature Cited

   Bender, B., Wiestler, O.D., and von Deimling, A. 1994. A device for processing large acrylamide gels. BioTechniques 16:204‐206.
   Budowle, B., Chakraborty, R., Giusti, A., Eisenberg, A., and Allen, R. 1991. Analysis of the VNTR locus D1S80 by PCR followed by high‐resolution PAGE. Am. J. Hum. Genet. 48:137‐144.
   Church, G. and Kieffer‐Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185‐188.
   Dib, C., Faure, S., Fizames, C., Samson, D., Drouot, N., Vignal, A., Millasseau, P., Marc, S., Hazan, J., Seboun, E., Lathrop, M., Gyapay, G., Morissette, J., and Weissenbach, J. 1996. A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature 380:152‐154.
   Dietrich, W., Katz, H., Lincoln, S.E., Shin, H.‐S., Friedman, J., Dracopoli, N.C., and Lander, E.S. 1992. A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131:423‐447.
   Hudson, T.J., Engelstein, M., Lee, M.K., Ho, E.C., Rubenfield, M.J., Adams, C.P., Housman, D.E., and Dracopoli, N.C. 1992. Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms. Genomics 13:622‐629.
   Lincoln, S.E., Daly, M.J., Lander, E.S. 1991. PRIMER: A Computer Program for Automatically Selecting PCR Primers. Version 0.5 Manual. MIT Center for Genome Research and Whitehead Institute for Biomedical Research, Cambridge, Mass.
   Vignal, A., Gyapay, G., Hazan, J., Nguyen, S., Dupraz, C., Cheron, N., Becuwe, N., Trachant, M., and Weissenbach, J. 1993. A nonradioactive multiplex procedure for genotyping of microsatellite markers. In Methods in Molecular Genetics, Vol. 1. Gene and Chromosome Analysis: Part A. (K.W. Adolph, ed.) pp. 211‐221. Academic Press, San Diego.
   Weissenbach, J., Gyapay, G., Dib, C., Vignal, A., Morissette, J., Millasseau, P., Vaysseix, G., and Lathrop, M. 1992. A second‐generation linkage map of the human genome. Nature 359:794‐801.
Key Reference
   Weber, J.L. and May, P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44:388‐396.
  Seminal paper describing the length polymorphism of SSLP, their detection by PCR, and their use as a rich pool of genetic markers.
   Vignal et al., 1993. See above.
  First published report of the use of nonradioactive multiplex methods for genotyping microsatellite markers.
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