Manual Methods for PCR‐Based Forensic DNA Analysis

David H. Bing1, Fredrick R. Bieber2

1 Genomics Collaborative, Cambridge, Massachusetts, 2 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.6
DOI:  10.1002/0471142905.hg1406s19
Online Posting Date:  May, 2001
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Abstract

This unit provides validated PCR‐based methods to test for sequence polymorphisms and length polymorphisms. It includes a description of the reverse dot blot method for detecting sequence polymorphisms. The forensic PCR systems used to detect length polymorphisms are based on detection of different‐sized PCR products following electrophoresis in either native or denaturing polyacrylamide gels. The unit offers a description of the amplification and detection of the variable number of tandem repeat (VNTR) length polymorphism at the D1S80 locus. It also describes the multiplex amplification of three separate autosomal short tandem repeats (STRs) and the X‐ and Y‐linked amelogenin alleles. These PCR products are electrophoresed on a denaturing polyacrylamide gel. Support protocols for creating permanent records of silver‐stained gels, checking the quality of reagents, and interpreting PCR‐based tests are provided.

     
 
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Table of Contents

  • Basic Protocol 1: AmpliTYPE PM+DQA1 Polymorphic Loci PCR Amplification and Typing
  • Basic Protocol 2: AmpliFLP D1S80 Locus VNTR PCR Amplification and Typing
  • Basic Protocol 3: PCR Amplification and Typing of CFSPO1, TPOX and, THO1 (Short Tandem Repeat) Loci and Amelogenin Locus
  • Support Protocol 1: Silver Staining of Polyacrylamide Gels with the Promega DNA Silver Staining System
  • Support Protocol 2: Creation of a Permanent Record for Silver‐Stained Gels Using DNA Electrophoresis Duplicating Film
  • Support Protocol 3: SYBR Green Staining of Polyacrylamide Gels
  • Support Protocol 4: Quality Control for Critical PCR‐Based Forensic Genetics Reagents
  • Support Protocol 5: Guidelines for Interpretation of PCR‐Based Forensic Genetic Typing Results
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: AmpliTYPE PM+DQA1 Polymorphic Loci PCR Amplification and Typing

  Materials
  • AmpliType PM+DQA1 PCR Amplification and Typing Kit (Perkin‐Elmer Applied Biosystems) containing:
    •   100 ng/ml PM+DQA1 control DNA
    •   AmpliType PM PCR reaction mix
    •   AmpliType PM primer set (this also contains the DQA1 primer set)
    •   Mineral oil
    •   AmpliType PM and DQA1 DNA probe strips
    •   Enzyme conjugate: horseradish peroxidase–strepavidin (HRP‐SA)
    •    recipeChromogen: 3,3′,5,5′‐tetramethylbenzidine (TMB; see recipe for reconstitution and storage)
  • DNA extracted from evidence samples (unit 14.3) containing 0.5 to 5 ng DNA
  • Reagent blank: sample extracted by same procedure used on evidence samples, but containing no DNA sample (see )
  • 200 mM disodium EDTA, pH 8.0 ( appendix 2D; store up to 3 months at room temperature)
  • NuSieve and SeaKem GTG agarose (FMC Bioproducts)
  • recipe0.5× TBE buffer (see recipe) with and without 0.5 µg/ml ethidium bromide
  • recipe5× loading solution (see recipe)
  • 50 ng/µl 123‐bp DNA ladder (Life Technologies)
  • recipeHybridization solution (see recipe), room temperature and prewarmed to 55°C
  • recipeWash solution (see recipe), prewarmed to 55°C (±1°C)
  • recipeCitrate buffer (see recipe)
  • recipe3% hydrogen peroxide (see recipe)
  • Model 480 thermal cycler, GeneAmp PCR System 9600 (both from Perkin‐Elmer Applied Biosystems), or equivalent
  • GeneAmp thin‐walled reaction tubes (for Model 480 cycler) or MicroAmp tubes with caps (for GeneAmp PCR System 9600)
  • Micropipettor with hydrophobic filter–plugged tips (Rainin)
  • Model H5 or H4 electrophoresis apparatus for horizontal submarine electrophoresis (Life Technologies) or equivalent electrophoresis apparatus
  • Model 4001 electrophoresis power supply (Life Technologies) or equivalent power supply capable of providing up to 2000 V
  • HCT Shaker Plus Model 7746 Sci Art heating shaking water bath (Bellco Biotechnology) or equivalent, set to 55°C
  • Calibrated total‐immersion thermometer (Cole‐Parmer)
  • AmpliType DNA typing tray (Perkin‐Elmer Applied Biosystems)
  • Glass flasks
  • Thermolyne Rotomix Model TY 50800 orbital shaker (Fisher) or equivalent
  • Polaroid camera with Type 52 (black and white) film, Polaroid one‐step MPX photographic copier with T1900 color film, or any suitable camera setup
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and interpretation of genetic typing results (see protocol 8)
NOTE: As with all PCR methodology, exercise great care to ensure a lack of contamination of the preparations with unwanted DNA. A separate area should be reserved for setting up PCR (see appendix 2D for further discussion of special considerations for PCR experiments).

Basic Protocol 2: AmpliFLP D1S80 Locus VNTR PCR Amplification and Typing

  Materials
  • AmpliFLP D1S80 PCR Amplification Kit (Perkin‐Elmer Applied Biosystems) containing:
    •   D1S80 PCR reaction mix
    •   5 mM MgCl 2
    •   Mineral oil
    •   Control DNA 3 (D1S80 type 18,31)
    •   D1S80 allelic ladder (27 amplified alleles; 14 and 16 through 31)
  • DNA extracted from evidence samples (unit 14.3) containing 0.5 to 5 ng DNA
  • Reagent blank: sample extracted by the same procedure used on evidence samples, but containing no DNA sample (see )
  • 95% ethanol
  • recipe10× and 0.5× TBE buffer (see recipe)
  • GeneAmp Detection Gel High‐Resolution Gel Concentrate and Loading Buffer Kit (Perkin‐Elmer Applied Biosystems) including:
    •   GeneAmp detection gel high‐resolution gel concentrate
    •   GeneAmp detection gel loading buffer
  • 10% ammonium persulfate (prepare fresh on day of use)
  • TEMED
  • Petrolatum
  • 40% (v/v) methanol
  • 160 mM nitric acid (HNO 3; add 13.3 ml of 6 N HNO 3 to 487 ml H 2O and mix thoroughly; store in Nalgene carboy or glass bottles up to 3 months at room temperature)
  • 12 mM silver nitrate (AgNO 3; prepare fresh on day of use)
  • 37% formaldehyde
  • recipeDeveloper solution for silver staining (see recipe), 2° to 8°C
  • Stop solution: 100 mM citric acid (prepare from 1 M stock in sterile H 2O; store sterile stock up to 1 month at room temperature)
  • Model 480 thermal cycler, GeneAmp PCR System 9600 (both from Perkin‐Elmer Applied Biosystems), or equivalent
  • GeneAmp thin‐walled reaction tubes (for Model 480 cycler) or MicroAmp tubes with caps (for GeneAmp PCR System 9600)
  • Dedicated positive‐displacement pipettor or micropipettor with hydrophobic filter–plugged tips
  • Model SA‐32 electrophoresis apparatus for vertical polyacrylamide electrophoresis (Life Technologies) or equivalent
  • GelBond PAG film (FMC Bioproducts)
  • Glue stick or glycerol
  • Gel‐sealing tape
  • 60‐ml syringe (optional)
  • Model 4001 electrophoresis power supply (Life Technologies) or equivalent power supply capable of providing up to 2000 V
  • Gel loading capillary pipet tips
  • 380 × 255 × 50–mm staining dishes (e.g., Pyrex glass baking dishes)
  • Thermolyne Rotomix Model TY 50800 orbital shaker (Fisher) or equivalent
  • Additional reagents and equipment for gel photography (see protocol 1) or preparing permanent record of silver‐stained gels (see protocol 5) and interpretation of genetic typing results (see protocol 8)
CAUTION: The GeneAmp detection gel (GDG) solution is a poisonous liquid. It is harmful if swallowed or absorbed through skin. Avoid contact with eyes, skin and clothing. Wear lab gloves, safety glasses and lab coat when handling GDG. Wash hands thoroughly after use.NOTE: As with all PCR methodology, exercise great care to ensure a lack of contamination of the preparations with unwanted DNA. A separate area should be reserved for setting up PCR (see appendix 2D for further discussion of special considerations for PCR experiments).

Basic Protocol 3: PCR Amplification and Typing of CFSPO1, TPOX and, THO1 (Short Tandem Repeat) Loci and Amelogenin Locus

  Materials
  • Amelogenin STR System (Promega) including:
    •  Amelogenin 10× primer pair
    •  STR 10× buffer
    •  Positive control DNA
    •  2× STR loading dye
    •  Amelogenin standard
    •  pGEM DNA markers
  • GenePrint CTT STR System (Promega) including:
    •  CSF1PO‐TPOX‐THO1 10× multiplex primer pair mix
    •  STR 10× buffer
    •  Positive control DNA
    •  2× STR loading dye
    •  CTT allelic ladder
    •  pGEM DNA markers
  • 5 U/ml Taq DNA polymerase (e.g., Perkin‐Elmer Applied Biosystems)
  • 8 mg/ml bovine serum albumin (Sigma; BSA fraction V; optional; store in 50‐µl single‐use aliquots at –20°C; thaw at room temperature )
  • DNA extracted from evidence samples (unit 14.3) containing up to 5 ng DNA in ≤5µl
  • 0.5% (v/v) acetic acid in 95% methanol (store up to 1 day at 28° to 30°C)
  • Bind‐Silane (Pharmacia Biotech)
  • Gel Slick solution (FMC Bioproducts)
  • recipe4% denaturing PAGE stock (see recipe)
  • 10% ammonium persulfate (prepare fresh on day of use)
  • TEMED
  • recipe0.5× TBE buffer (see recipe)
  • GeneAmp PCR System 9600 (Perkin‐Elmer Applied Biosystems) or equivalent
  • MicroAmp reaction tubes with caps (Perkin‐Elmer Applied Biosystems)
  • Model SA‐32 electrophoresis apparatus for vertical polyacrylamide electrophoresis (Life Technologies) or equivalent
  • 4‐mm spacers
  • 100‐ml squeeze bottle
  • Bulldog clamps
  • 10‐ to 50‐ml syringe
  • Model 4001 electrophoresis power supply (Life Technologies) or equivalent power supply capable of providing up to 2000 V
  • 94°C heating block
  • Additional reagents and equipment for silver staining (see protocol 2, steps to , or see protocol 4) or staining with SYBR Green (see protocol 6) and interpretation of genetic typing results (see protocol 8)
NOTE: As with all PCR methodology, exercise great care to ensure a lack of contamination of the preparations with unwanted DNA. A separate area should be reserved for setting up PCR (see appendix 2D for further discussion of special considerations for PCR experiments).

Support Protocol 1: Silver Staining of Polyacrylamide Gels with the Promega DNA Silver Staining System

  Materials
  • D1S80 or CTAT gel (see Basic Protocols protocol 22 and protocol 33)
  • DNA Silver Staining System (Promega) including:
    •  37% formaldehyde
    •  Silver nitrate
    •  10 mg/ml sodium thiosulfate
    •  Sodium carbonate
  • Fix/stop solution: mix 100 ml glacial acetic acid and 900 ml distilled H 2O
  • Orbital shaker

Support Protocol 2: Creation of a Permanent Record for Silver‐Stained Gels Using DNA Electrophoresis Duplicating Film

  Materials
  • Silver‐stained gel (see protocol 2, steps to , or see protocol 4)
  • DNA Electrophoresis Duplicating Film (EDF; Promega)
  • Previously developed piece of Kodak X‐OMat AR film
  • Automatic film processor

Support Protocol 3: SYBR Green Staining of Polyacrylamide Gels

  Materials
  • Gel (see Basic Protocols protocol 22 and protocol 33), prepared without GelBond
  • SYBR Green I nucleic acid gel stain (FMC Bioproducts)
  • recipe0.5× TBE (see recipe)
  • 50‐ml conical polypropylene tube
  • Polaroid camera and Type 57 film
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Figures

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Literature Cited

Literature Cited
   Allen, R., Budowle, B., Chakraborty, R., Giusti, A., and Eisenberg, A. 1991. Analysis of the VNTR locus AmpliFLP D1S80 by the PCR followed by high‐resolution PAGE. Am. J. Hum. Genet. 48:137‐144.
   Bodmer, J.G., Marsh, S.G.E., Albert, E., Bodmer, W.F. Dupont, B., Erlich, H.A., Mach, B., Mayr, W.R., Parham, P., Sasasuki, T., Schreuder, G.M.T., Strominger, J.L., Svejgaard, A., Terassaki, P.I. 1992. Nomenclature for factors of the HLA system, 1991. In HLA 1991 (T. Tsuji M. Aizawa and T. Sasasuki eds.) pp. 17‐31. Oxford University Press Tokyo.
   Budowle, B., Baechtel, F.S., Comey, C.T. 1994. Some considerations for use of AMP‐FLPs for identity testing. In Advances in Forensic Haemogenetics,vol. 4 (C.H. Rittner and P.M. Schneider, eds.) pp. 11‐17. Springer Verlag, New York.
   Budowle, B., Lindsey, J.A., DeCou, J.A., Koons, B.W., Giusti, A.M., and Comey, C.T. 1995. Validation and population studies of the loci LDLR,GYPA, HBGG, D7S8, and GC (PM loci), and HLA‐DQα using a multiplex amplification and typing procedure. J. Forensic Sci. 40:45‐50.
   Comey, C.T. and Budowle, B. 1991. Validation studies on the analysis of the HLA‐DQα locus using the polymerase chain reaction. J. Forensic Sci 36:1633‐1648.
   Comey, C.T., Budowle, B., Adams, D.E., Baumstark, A.L., Lindsey, J.A., and Presley, L.A. 1993. PCR amplification and typing of the HLA DQα gene in forensic samples. J. Forensic Sci. 38:239‐249.
   Cosso, S. and Reynolds, R. 1995. Validation of the D1S80 PCR amplification kit for forensic casework analysis according to TWGDAM guidelines. J. Forensic Sci. 40:424‐434.
   Crouse, C.A. and Schumm, J. 1995. Investigation of species specificity using nine PCR‐based human STR systems. J. Forensic Sci. 40:952‐956.
   Crouse, C.A., Vincek, V., and Caraballo, B.K. 1994. Analysis and interpretation of the HLA DQα “1.1 weak‐signal” observed during the PCR‐based typing method. J. Forensic Sci. 39:41‐51.
   Crouse, C.A., Nippes, D.C., and Ritzline, E.L. 1996. Confirmation of PM typing protocols for consistent and reliable results. J. Forensic Sci. 41:493‐496.
   FBI, 1994. PCR‐Based Typing Protocols,1st edition. FBI Laboratory, Washington, D.C.
   FBI. 1996. PCR‐Based Typing Protocols,2nd edition FBI Laboratory, Washington, D.C.
   Fildes, N. and Reynolds, R. 1995. Consistency and reproducibility of AmpliType PM results between seven laboratories: Field trial results. J. Forensic Sci. 40:279‐286.
   Hammond, H.A., Jin, L., Zhong, Y., Caskey, C.T., Chakraborty, R. 1994. Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am. J. Hum. Genet. 55:175‐189.
   Kasai, K., Nakamura, Y., and White, R. 1990. Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science. J. Forensic Sci. 35:1196‐1200.
   Lins, A.M., Sprecher, C.J., Puers, C., and Schumm, J.W. 1996. Multiplex set for amplification of polymorphic short tandem repeat loci: Silver stain and fluorescent detection. BioTechniques 20:882‐889.
   Micka, K.A., Sprecher, C.J., Lins, A.M., Theisen Comey, C., Koons, B.W., Crouse, C., Endean, D., Pirelli, K., Lee, S.B., Duda, N., Ma, M., and Schumm, J.W. 1996. Validation of multiplex polymorphic STR amplification sets developed for personal identification applications. J. Forensic Sci. 41:582‐590.
   Nakamura, Y., Carlson, M., Krapcho, K., and White, R. 1988. Isolation and mapping of a polymorphic DNA sequence (pMCT118) on chromosome 1p (D1S80). Nucl. Acids Res. 16:9364.
   Reynolds, R., Sensabaugh, G., and Blake, E. 1991. Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction. Anal. Chem. 63:2‐15.
   Saiki, R.K., Walsh, P.S., Levenson, C.H., and Erlich, H.A. 1989. Genetic analysis of amplified DNA with immobilized sequence‐specific oligonucleotide probes. Proc. Natl. Acad. Sci. U.S.A. 86:6230‐6234.
   Sensabaugh, G.F. and Blake, E.T. 1993. DNA analysis in biological evidence: Applications of the polymerase chain reaction. In Forensic Science Handbook,Vol. 3 (R. Saferstein, ed.) pp. 416‐452. Reagents/Prentice Hall, Englewood Cliffs, N.J.
   Sprecher, C.J., Puers, C., Lins, A.M., Schumm, J.W. 1996. A general approach to analysis of polymorphic short tandem repeat loci. BioTechniques 20:266‐276.
   Technical Working Group on DNA Analysis Methods (TWGDAM). 1995. Guidelines for a quality assurance program for DNA analysis. Crime Lab. Digest 22:21‐43.
   Word, C.J., Sawosik, T.M., and Bing, D.H. 1997. Summary of validation studies from twenty‐six laboratories in the United States and Canada on the use of the AmpliType PM PCR. J. Forensic Sci. 42:39‐48.
Key References
   Sensabaugh and Blake, 1993. See above.
  General reference on the use of PCR based DNA testing for forensic testing.
   Inman, K. and Rudin, N. 1997. An Introduction to Forensic DNA Analysis. CRC Press, Boca Raton, Fla.
  Provides an overview of DNA‐based forensic genetic testing; includes an introduction to human genetics, general descriptions of the molecular basis for validated DNA based forensic tests, examples of cases where the techniques have been used, and a summary of the legal review of the technology.
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