RFLP Analysis of Forensic DNA Samples with Single‐Locus VNTR Genetic Markers

David H. Bing1, Frederick R. Bieber2

1 Genomics Collaborative, Cambridge, Massachusetts, 2 Brigham and Woman's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.5
DOI:  10.1002/0471142905.hg1405s18
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Resolution of Restriction‐Digested Forensic DNA Samples for RFLP Analysis
  • Basic Protocol 2: Downward Transfer Southern Blotting of Forensic DNA Samples for RFLP Analysis
  • Basic Protocol 3: Hybridization and Development of Southern Blot with Chemiluminescent Labeled VNTR Probes
  • Support Protocol 1: Blot‐Stripping Procedure for Chemiluminescent Protocols used with VNTR Probes
  • Support Protocol 2: Quality Control Procedures for Forensic RFLP Analysis
  • VNTR Probe
  • Reagents and Solutions
  • Commentary
  • Acknowledgement
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Resolution of Restriction‐Digested Forensic DNA Samples for RFLP Analysis

  Materials
  • DNA samples extracted by organic method from liquid blood or stains (unit 14.3)
  • 10 U/µl HaeIII restriction endonuclease and HaeIII restriction buffer concentrate (Life Technologies)
  • 7.0 M ammonium acetate
  • Absolute ethanol (−20°C) and 70% ethanol (room temperature)
  • TE buffer, pH 8.0 ( appendix 2D)
  • DNA‐typing‐grade agarose (FMC Bioproducts)
  • recipe1× TAE buffer (see recipe)
  • Visual marker: adenovirus 2 KpnI digest (Life Technologies)
  • 5 mg/ml and 0.5 µg/ml ethidium bromide ( appendix 2D)
  • HaeIII‐digested DNA from K562 cell line (Life Technologies, Lifecodes, Promega, or National Institute of Standards and Technology, Washington, D.C.)
  • 22‐ to 500‐bp DNA molecular size standards (Life Technologies, Lifecodes, or Promega)
  • recipe5× loading solution (see recipe)
  • 56°C water bath (Bellco heating shaking water bath or equivalent)
  • Gel‐casting apparatus for 11 × 14–cm agarose gel (unit 2.7) with 16‐well comb
  • Apparatus for horizontal submarine electrophoresis and power supply for 2000 V ( Life Technologies or equivalent; also see unit 2.7)
  • Polaroid no. 553 film (ASA 400) and red filter
  • Additional reagents and equipment for minigel analysis of DNA (e.g., CPMB UNIT ) and agarose gel electrophoresis of DNA and gel photography (unit 2.7)

Basic Protocol 2: Downward Transfer Southern Blotting of Forensic DNA Samples for RFLP Analysis

  Materials
  • Restriction‐digested forensic samples on analytical gel
  • Denaturing solution: 0.5 M NaOH/1.5 M NaCl
  • Neutralizing solution: 0.5 M Tris⋅Cl, pH 7.5 ( appendix 2D)/1.5 M NaCl
  • 10× and 2× SSC, pH 7.0 ( appendix 2D)
  • Rubbermaid plastic boxes large enough to accommodate gel
  • Orbital shaker
  • Opititran BA‐S or Nytran 0.2 µm‐pore‐size nylon membrane (Schleicher & Schuell)
  • No. 2 pencil
  • TurboBlotter Downward Transfer System (Schleicher & Schuell) including:
  •   Stack tray
  •   GB004 and GB002 precut blotting paper
  •   Buffer tray
  •   Buffer wick and wick cover
  • 80°C drying oven
  • Stratalinker 2400 (Stratagene) or equivalent UV cross‐linker

Basic Protocol 3: Hybridization and Development of Southern Blot with Chemiluminescent Labeled VNTR Probes

  Materials
  • Hybridized membranes containing digested forensic DNA (see protocol 2)
  • recipeACES 2.0 hybridization solution (Life Technologies; also see recipe)
  • Alkaline phosphatase–labeled probes for VNTR alleles (Life Technologies, Lifecodes, or Promega)
  • Visual marker probe for adenovirus 2 KpnI digest (Life Technologies)
  • recipe1× and 0.5× ACES 2.0 wash I solution (Life Technologies; also see recipe)
  • recipe1× ACES 2.0 final wash buffer (Life Technologies; also see recipe)
  • Lumi‐Phos Plus (Life Technologies)
  • Rubbermaid plastic tubs
  • 55°C shaking water bath
  • Orbital shaker
  • Whatman 3MM chromatography paper
  • Platform rocker
  • Blunt‐end forceps
  • Plastic folders (Lifecodes)
  • Impulse Sealer (Bellco Biotechnology)
  • Kodak X‐Omat RP film
  • X‐ray film cassettes

Support Protocol 1: Blot‐Stripping Procedure for Chemiluminescent Protocols used with VNTR Probes

  Materials
  • Probed membrane packet (see protocol 3)
  • recipe1× stripping solution (see recipe)
  • SSC ( appendix 2D)
  • Blunt‐end forceps
  • Rubbermaid plastic tubs
  • 65°C rotating environmental shaker or shaking water bath
  • Orbital shaker
  • Whatman 3MM chromatography paper
  • Whatman 3MM paper folders
  • Zip‐lock plastic bags

Support Protocol 2: Quality Control Procedures for Forensic RFLP Analysis

  Materials
  • 0.50 µg/µl intact ΦX174 DNA (Life Technologies) in TE buffer, pH 8.0 ( appendix 2D)
  • 10 U/µl HaeIII restriction endonuclease and HaeIII buffer concentrate (Life Technologies)
  • 1 U/µl HaeIII restriction endonuclease: dilute 10 U/µl HaeIII to 1 U/µl with TE buffer, pH 8.0 ( appendix 2D)
  • 7.0 M ammonium acetate
  • Absolute ethanol (−20°C) and 70% ethanol (room temperature)
  • TE buffer, pH 8.0
  • 1.5% agarose gel (25 ml, ∼8 × 7–cm, with at least 8 wells) prepared with molecular biology–grade agarose in recipe1× TAE buffer (see recipe) containing 0.5 µg/ml ethidium bromide (see unit 2.7 and CPMB UNIT )
  • 0.25µg/µl HaeIII‐predigested ΦX174 DNA (Life Technologies) in TE buffer, pH 8.0 ( appendix 2D)
  • recipeTAE buffer (see recipe)
  • Additional reagents and equipment for agarose gel electrophoresis and gel photography (unit 2.7 and CPMB UNIT )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adams, D.E., Presley, L.A., Baumstark, A.L., Henstey, K.W., Hill, A.L., Aroe, K.S., Campbell, P.A., McLaughlin, C.M., Budowle, B., Giusti, A.M., Smerick, J.B., and Baechtel, F.S. 1991. Deoxyribonucleic acid (DNA) analysis by restriction fragment length polymorphisms of blood and other body fluid stains subjected to contamination and environmental insults. J. Forensic Sci. 36:1284‐1296.
   Bing, D.H. 1997. Development of DNA typing methods for forensic analysis. In More Chemistry in Crime (S.M. Gerber and R. Saferstein, eds.) pp. 33‐57. American Chemical Society, Washington, D.C.
   Budowle, B. and Monson, K.L. 1993. The forensic significance of various reference population databases for estimating the rarity of variable number of tandem repeat (VNTR) loci profiles. EXS 67:177‐191.
   Budowle, B., Waye, J.S., Shutler, G.G., and Baechtel, F.S. 1990. HaeIII: A suitable restriction endonuclease for restriction fragment length polymorphism analysis of biological samples. J. Forensic Sci. 35:530‐536.
   Budowle, B., Giusti, A.M., Waye, J.S., Baechtel, F.S., Fourney, R.M., Adams, D.E., Presley, L.A., Deadman, H.A., and Monson, K.L. 1991. Fixedbin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons. Am. J. Hum. Genet. 48:841‐855.
   Budowle, B., Baechtel, F.S., Comey, C.T., Giusti, A.M., and Klevan, L. 1995. Simple protocols for typing forensic biological evidence: Chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RFLP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms. Electrophoresis 16:1559‐1567.
   FBI. 1990. FBI Laboratory Procedures for the Detection of Restriction Fragment Length Polymorphisms in Human DNA (available upon request from Federal Bureau of Investigation).
   Johnson, E.D., Kotowoski, T.M., and McCaffery 1996. Chemiluminescent detection of RFLP patterns in forensic DNA analysis. J. Forensic. Sci. 41:569‐578.
   National Research Council. 1996. The evaluation of forensic DNA evidence. National Academy Press, Washington, D.C.
   Technical Working Group on DNA Analysis Methods (TWGDAM) 1995. Guidelines for a quality assurance program for DNA analysis. Crime Laboratory Digest 22:21‐43.
   Waye, J.S. and Fourney, R.M. 1993. Forensic DNA typing of highly polymorphic VNTR loci. In Forensic Science Handbook, vol.III (R. Saferstein, ed.) pp 358‐397. Prentice Hall, Englewood Cliffs, N.J.
Key References
   Klevan, L., Horton, L., Carlson, D.P., and Eisenberg, A.J. 1995. Chemiluminescent detection of DNA probes in forensic analysis. Electrophoresis 16:1553‐1558.
  Review of the scientific basis and experimental results with chemiluminescent detection methodology in the analysis of forensic samples.
   Waye and Fourney, 1993. See above.
  Review article that describes in detail procedures and protocols developed for RFLP analysis of forensic samples with VNTR markers.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library