Short Tandem Repeat Analysis for Human Identity Testing

John M. Butler1

1 National Institute of Standards and Technology, Gaithersburg, MD
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.8
DOI:  10.1002/0471142905.hg1408s41
Online Posting Date:  September, 2004
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Abstract

Short tandem repeat (STR) typing has become the workhorse of modern forensic DNA analysis. The most common form of STR typing uses laser‐induced fluorescence detection of dye‐labeled polymerase chain reaction (PCR) products following capillary electrophoresis (CE) size‐based separation. This unit describes the techniques and marker systems most widely used around the world in constructing criminal DNA databases and conducting forensic casework. In addition, these same STR loci and multiplex PCR assays are commonly used for paternity testing, missing persons, and mass disaster investigations. Both autosomal and Y‐chromosome STRs are discussed in the context of their various applications. Interpretation issues surrounding degraded or mixed DNA specimens as well as tri‐allelic patterns and variant alleles are also illustrated. Finally, future technologies and DNA marker systems are briefly explored relative to the state‐of‐the‐science today.

Keywords: human identity testing; forensic DNA typing; short tandem repeat; STR; genotyping; PCR; ABI 310; ABI 3100; commercial STR kits

     
 
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Table of Contents

  • Basic Protocol 1: Amplification of STR Markers: Identifiler Kit
  • Basic Protocol 2: Separation and Detection of STR Amplicons: Use of ABI 310 Genetic Analyzer
  • Basic Protocol 3: Data Analysis and STR Genotyping
  • Alternate Protocol 1: Amplification of STR Markers: Powerplex 16 Kit
  • Alternate Protocol 2: Amplification of Y‐STR Markers: Y‐PLEX 12 Kit
  • Alternate Protocol 3: Separation and Detection of STR Amplicons: Use of Multi‐Capillary ABI 3100 System
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amplification of STR Markers: Identifiler Kit

  Materials
  • Identifiler STR kit (Applied Biosystems) containing:
    • AmpliTaq Gold
    • Primer mix
    • PCR reaction mix
    • Control DNA 9947A
  • DNA extract of appropriate biological specimens that have been previously quantified, and accompanying positive and negative controls (unit 14.3)
  • Thermal cycler (e.g., GeneAmp 9600 or 9700 in 9600‐emulation mode)
  • 200‐µl thin‐walled PCR tubes
  • 1.5‐ml sterile tube

Basic Protocol 2: Separation and Detection of STR Amplicons: Use of ABI 310 Genetic Analyzer

  Materials
  • Methanol or isopropanol wipes
  • POP‐4 Performance Optimized Polymer (Applied Biosystems)
  • 10× Genetic Analyzer buffer (Applied Biosystems)
  • Hi‐Di formamide (Applied Biosystems)
  • Matrix standard set DS‐33 (6‐FAM, VIC, NED, PET, and LIZ; Applied Biosystems)
  • PCR‐amplified samples (Identifiler sample amplified; see protocol 1)
  • GeneScan‐500 LIZ size standard (Applied Biosystems)
  • Allelic ladder from the Identifiler kit (see protocol 1)
  • Genetic Analyzer (ABI Prism 310)
  • Computer with ABI 310 data collection software (version 3.0.0)
  • Kimwipes
  • Capillaries (47 cm × 50‐µm i.d.; Applied Biosystems)
  • 1.0‐ml glass syringe (Applied Biosystems)
  • 15‐ml Falcon tube
  • 4‐ml Genetic Analyzer buffer vials (Applied Biosystems)
  • 48‐tube sample tray (Applied Biosystems)
  • 1.5‐ml sterile tube
  • 0.5‐ml Genetic Analyzer sample tubes (Applied Biosystems)
  • Genetic Analyzer septa for 0.5 ml sample tubes (Applied Biosystems)

Basic Protocol 3: Data Analysis and STR Genotyping

  Materials
  • GeneScan software version 3.7 (Applied Biosystems)
  • Genotyper NT software version 3.7 (Applied Biosystems)
  • GeneScan reference guide

Alternate Protocol 1: Amplification of STR Markers: Powerplex 16 Kit

  Materials
  • PowerPlex 16 STR kit (Promega) which includes:
    • Primer mix
    • PCR components such as dNTPs, MgCl 2, and 10× Gold StaR buffer
  • AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • DNA extract of appropriate biological specimens that have been previously quantified, and accompanying positive and negative controls (unit 14.3)
  • 0.20‐ml thin‐walled PCR tubes
  • Thermal cycler (e.g., GeneAmp 9600 or 9700 in 9600‐emulation mode)

Alternate Protocol 2: Amplification of Y‐STR Markers: Y‐PLEX 12 Kit

  Materials
  • Y‐PLEX 12 kit (ReliaGene Technologies) which includes:
    • Primer mix
    • PCR components such as dNTPs, MgCl 2, and 10× buffer
  • AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • DNA extract of appropriate biological specimens that have been previously quantified, and accompanying positive and negative controls (unit 14.3)
  • 0.20‐ml thin‐walled PCR tubes
  • Thermal cycler (e.g., GeneAmp 9600 or 9700 in 9600‐emulation mode)

Alternate Protocol 3: Separation and Detection of STR Amplicons: Use of Multi‐Capillary ABI 3100 System

  Materials
  • 3100 POP‐4 or 3700 POP‐6 polymer (Applied Biosystems)
  • Matrix standards, DS‐33 for G5 filter (Applied Biosystems)
  • Hi‐Di formamide (Applied Biosystems)
  • PCR samples amplified in protocol 1Basic Protocol, protocol 4, or protocol 5
  • GeneScan‐500 LIZ size standard (Applied Biosystems)
  • GS500 ROX size standard (for Y‐PLEX 12)
  • ILS600 CXR size standard (for PowerPlex 16)
  • ABI Prism 3100 Genetic Analyzer
  • Computer with 3100 Genetic Analyzer data collection software (version 1.0.1)
  • Centrifuge
  • ABI 3100 capillary array (36 cm; Applied Biosystems)
  • 96‐well plate (Applied Biosystems)
  • Plate septum (Applied Biosystems)
  • Plate base (Applied Biosystems)
  • Plate retainer (Applied Biosystems)
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Figures

Videos

Literature Cited

   Applied Biosystems. 2000. GeneScan Reference Guide: Chemistry Reference for the ABI Prism 310 Genetic Analyzer. Foster City, CA.
   Applied Biosystems. 2001a. AmpFlSTR Identifiler PCR Amplification Kit User's Manual. Foster City, CA.
   Applied Biosystems. 2001b. ABI Prism 310 Genetic Analyzer User's Manual. Foster City, CA.
   Applied Biosystems. 2001c. ABI Prism 3100 Genetic Analyzer User's Manual. Foster City, CA.
   Applied Biosystems. 2001d. ABI Prism Genotyper 3.7 NT Software User's Manual. Foster City, CA.
   Belgrader, P., Smith, J.K., Weedn, V.W., and Northrup, M.A. 1998. Rapid PCR for identity testing using a battery‐powered miniature thermal cycler. J.Forensic Sci. 43:315‐319.
   Budowle, B., Smith, J., Moretti, T., and DiZinno, J. 2000. DNA Typing Protocols: Molecular Biology and Forensic Analysis. Eaton Publishing, Natick, MA.
   Butler, J.M. 2001. Forensic DNA Typing: Biology and Technology behinds STR Markers. Academic Press, London.
   Butler, J.M. 2003. Recent developments in Y‐short tandem repeat and Y‐single nucleotide polymorphism analysis. Forensic Sci. Rev. 15:91‐111.
   Butler, J.M., Schoske, R., Vallone, P.M., Kline, M.C., Redd, A.J., and Hammer, M.F. 2002. A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci. Int. 129:10‐24.
   Butler, J.M., Shen, Y., and McCord, B.R. 2003. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci. 48:1054‐1064.
   Findlay, I., Taylor, A., Quirke, P., Frazier, R., and Urquhart, A. 1997. DNA fingerprinting from single cells. Nature. 389:555‐556.
   Gaines, M.L., Wojtkiewicz, P.W., Valentine, J.A., and Brown, C.L. 2002. Reduced volume PCR amplification reactions using the AmpFlSTR Profiler Plus kit. J. Forensic Sci. 47:1224‐1237.
   Gill, P. 2001. An assessment of the utility of single nucleotide polymorphisms (SNPs) for forensic purposes. Int. J. Legal Med. 114:204‐210.
   Gill, P. 2002. Role of short tandem repeat DNA in forensic casework in the UK—past, present, and future perspectives. BioTechniques. 32:366‐372.
   Krenke, B.E., Tereba, A., Anderson, S.J., Buel, E., Culhane, S., Finis, C.J., Tomsey, C.S., Zachetti, J.M., Masibay, A., Rabbach, D.R., Amiott, E.A., and Sprecher, C.J. 2002. Validation of a 16‐locus fluorescent multiplex system. J. Forensic Sci. 47:773‐785.
   Leclair, B., Sgueglia, J.B., Wojtowicz, P.C., Juston, A.C., Fregeau, C.J., and Fourney, R.M. 2003. STR DNA typing: Increased sensitivity and efficient sample consumption using reduced PCR reaction volumes. J. Forensic Sci. 48:1001‐1013.
   McCord, B. 2003. Troubleshooting capillary electrophoresis systems. Profiles in DNA (Promega Corporation) 6:10‐12. http://www.promega.com/profiles/602/ProfilesInDNA_602_10.pdf.
   Moretti, T.R., Baumstark, A.L., Defenbaugh, D.A., Keys, K.M., Smerick, J.B., and Budowle, B. 2001. Validation of short tandem repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J.Forensic Sci. 46:647‐660.
   National Institute of Justice (NIJ). 2000. The Future of Forensic DNA Testing: Predictions of the Research and Development Working Group of the National Commission on the Future of DNA Evidence. Washington, D.C. http://www.ojp.usdoj.gov/nij/pubs‐sum/183697.htm.
   Schoske, R., Vallone, P.M., Kline, M.C., Redman, J.W., and Butler, J.M. 2004. High‐throughput Y‐STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays. Forensic Sci. Int. 139:107‐121.
   Scientific Working Group on DNA Analysis Methods (SWGDAM). 2000. Short tandem repeat (STR) interpretation guidelines. Forensic Sci. Comm. 2(3);
   on‐line at http://www.fbi.gov/hq/lab/fsc/backissu/july2000/strig.htm.
   van Oorschot, R.A. and Jones, M.K. 1997. DNA fingerprints from fingerprints. Nature 387:767.
   Whitaker, J.P., Clayton, T.M., Urquhart, A.J., Millican, E.S., Downes, T. J., Kimpton, C.P., and Gill, P. 1995. Short tandem repeat typing of bodies from a mass disaster: High success rate and characteristic amplification patterns in highly degraded samples. BioTechniques. 18:670‐677.
Key References
   Butler, J.M. 2001 Forensic DNA Typing: Biology and Technology behinds STR Markers. Academic Press, London.
  Thorough description of STR typing process with illustrated examples.
Internet Resources
   http://www.cstl.nist.gov/biotech/strbase/
  STRBase Web site providing detailed information on STR markers used in human identity testing maintained by the author at NIST. A fairly comprehensive list of publications dealing with STRs and human identity testing is included.
   http://www.ystr.org
  Y‐STR Haplotype Reference Database containing information on core Y‐STR markers and haplotype frequencies observed in European, U.S., and Asian populations.
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