Isolation of DNA from Forensic Evidence

David H. Bing1, Frederick R. Bieber2, Mitchell M. Holland3, Edwin F. Huffine3

1 Genomics Cooperative, Cambridge, Massachusetts, 2 Brigham and Women's Hospital, Boston, Massachusetts, 3 The Armed Forces DNA Identification Laboratory, Rockville, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 14.3
DOI:  10.1002/0471142905.hg1403s26
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Phenol/Chloroform Extraction of DNA from liquid Blood and Dried Blood Stains
  • Basic Protocol 1: Organic Extraction of DNA from Blood for PCR Analysis
  • Basic Protocol 2: Organic Extraction of DNA from Blood for RFLP Analysis
  • Basic Protocol 3: Chelex Extraction of DNA from Blood Samples or Stains
  • Isolation of DNA from Blood Stains Prepared on FTA Gene Guard System
  • Basic Protocol 4: FTA Paper Isolation of DNA from Blood for RFLP Analysis
  • Basic Protocol 5: FTA Paper Isolation of DNA from Blood for PCR Analysis
  • Basic Protocol 6: Differential DNA Extraction from Sexual Assault Evidence
  • Support Protocol 1: Gram Modified Christmas Tree Stain for Detecting Spermatozoa
  • Alternate Protocol 1: Extraction of Nuclear DNA from Hair‐Root Samples for PCR Analysis
  • Alternate Protocol 2: Extraction of Mitochondrial DNA from Hair Shafts
  • Alternate Protocol 3: Extraction of DNA from Tissue Samples
  • Alternate Protocol 4: Extraction of DNA form Cigarette Butts
  • Alternate Protocol 5: Extraction of DNA from Bones
  • Alternate Protocol 6: Extraction of DNA from Teeth
  • Alternate Protocol 7: Chelex Extraction of DNA from Envelope Flaps and Stamps
  • Support Protocol 2: Yield‐Gel Determination of Quality and Quantity of Forensic DNA Samples
  • Support Protocol 3: Visualization of DNA in Yield Gels Using DAPI Staining
  • Support Protocol 4: Quantitation of DNA by Hybridization to Human DNA–Specific Probes
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Organic Extraction of DNA from Blood for PCR Analysis

  Materials
  • Blood‐stained material or whole blood sample
  • Blood from a known donor
  • recipeProtein lysis buffer (see recipe)
  • 20% (w/v) SDS, ultrapure ( appendix 2D)
  • recipe10 mg/ml proteinase K (see recipe)
  • recipeExtraction solutions I, recipeII, and recipeIII (see reciperecipes)
  • recipeH 2O‐saturated butanol (see recipe)
  • TE buffer, pH 8.0 ( appendix 2D) or Lifecodes dialysis buffer diluted 1:100
  • Centricon 100 concentrators (Amicon) and conical collection tubes
  • Refrigerated centrifuge with fixed‐angle rotor accommodating Centricon 100 concentrators

Basic Protocol 2: Organic Extraction of DNA from Blood for RFLP Analysis

  Materials
  • Blood‐stained sample or whole blood
  • recipeCell lysis buffer (see recipe), cold
  • recipeProtein lysis buffer (see recipe)
  • 20% (w/v) SDS, ultrapure ( appendix 2D)
  • recipe10 mg/ml proteinase K (see recipe)
  • 6 M sodium perchlorate
  • Additional reagents and equipment for extracting and concentrating DNA (as for PCR analysis; see protocol 1)

Basic Protocol 3: Chelex Extraction of DNA from Blood Samples or Stains

  Materials
  • Liquid blood sample or blood‐stained material
  • Blood from a known donor
  • recipe5% (w/v) Chelex suspension (see recipe; gently mix on magnetic stirrer to keep suspended during pipetting)
  • Microcentrifuge tubes with locking caps
  • Boiling water bath

Basic Protocol 4: FTA Paper Isolation of DNA from Blood for RFLP Analysis

  Materials
  • FTA Gene Guard System paper cards (Life Technologies or Promega)
  • Blood sample
  • FTA purification reagent (Life Technologies or Promega)
  • recipe10 mg/ml proteinase K (see recipe)
  • TE buffer, pH 8.0 ( appendix 2D)
  • 6‐mm paper punch
  • 2‐ml Spin‐EASE tubes (Life Technologies)
  • 60°C heating block

Basic Protocol 5: FTA Paper Isolation of DNA from Blood for PCR Analysis

  Materials
  • FTA Gene Guard System paper cards (Life Technologies or Promega)
  • Blood sample
  • FTA purification reagent (Life Technologies or Promega)
  • TE buffer, pH 8.0 ( appendix 2D)
  • 1‐mm or 3‐mm paper punch
  • 2‐ml Spin‐EASE tubes (Life Technologies)
  • 60°C heating block

Basic Protocol 6: Differential DNA Extraction from Sexual Assault Evidence

  Materials
  • Stained sample or swab
  • recipeProtein lysis buffer (see recipe)
  • 20% (w/v) SDS, ultrapure ( appendix 2D)
  • recipe10 mg/ml proteinase K (see recipe)
  • recipeExtraction solutions I, recipeII, and recipeIII (see reciperecipes)
  • recipeH 2O‐saturated butanol (see recipe)
  • TE buffer, pH 8.0 ( appendix 2D)
  • Saline: 0.9% (w/v) NaCl
  • 20% (w/v) Sarkosyl (N‐lauroylsarcosine; sterilize using 0.45‐µm filter and store up to 1 year at room temperature)
  • 100 mM EDTA, pH 7.3 ( appendix 2D)
  • 1 M dithiothreitol (DTT; appendix 2D)
  • 1.5‐ml Quick spin caps (Life Technologies)
  • Centricon 100 concentrators (Amicon) and conical collection tubes
  • Glass microscope slides

Support Protocol 1: Gram Modified Christmas Tree Stain for Detecting Spermatozoa

  Materials
  • 0.5% (w/v) crystal violet (Sigma; also called methyl violet 10B), aqueous
  • recipeGram's iodine (see recipe)
  • Acetone
  • recipeNuclear fast red stain (see recipe)
  • recipePicroindigocarmine stain (see recipe)
  • 95% ethanol
  • Microscope slides
  • 55°C circulating oven

Alternate Protocol 1: Extraction of Nuclear DNA from Hair‐Root Samples for PCR Analysis

  Materials
  • Sample hair to be tested
  • Pulled head hair from known donor
  • Shaft from each hair tested
  • recipe5% (w/v) Chelex suspension (see recipe; gently mix on magnetic stirrer to keep suspended during pipetting)
  • recipe10 mg/ml proteinase K (see recipe)
  • Scalpels
  • 56°C and boiling water bath

Alternate Protocol 2: Extraction of Mitochondrial DNA from Hair Shafts

  Materials
  • Hair specimen(s) (e.g., ∼2 cm in length or greater, mounted or loose)
  • Appropriate mounting medium solvent (e.g., toluene or 100% xylenes)
  • 10% (v/v) commercial bleach (7 mM sodium hypochlorite)
  • 100% ethanol
  • 5% (w/v) Terg‐A‐Zyme detergent (e.g., Baxter or VWR)
  • recipeExtraction buffer (see recipe)
  • 20 mg/ml proteinase K
  • 1 M dithiothreitol (DTT; appendix 2D)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • n‐butanol
  • TE buffer, pH 7.5 ( appendix 2D)
  • 1.7‐ml sterile microcentrifuge tubes
  • Micro tissue grinders (Fisher)
  • Sonicator
  • 56°C incubator (or 56°C heating block)
  • Centricon 30 concentrators (Amicon)
  • Centrifuge (e.g., IEC Clinical)
  • 15‐ml conical tubes (for large‐volume extractions)

Alternate Protocol 3: Extraction of DNA from Tissue Samples

  Materials
  • Cigarette butt to be tested
  • Cigarette butt from known donor
  • recipe5% Chelex suspension (see recipe; gently mix on magnetic stirrer to keep suspended during pipetting)
  • TE buffer, pH 8.0 ( appendix 2D)
  • Centricon 100 concentrators (Amicon) and conical collection tubes
  • Centrifuge with fixed‐angle rotor accommodating Centricon 100 concentrators

Alternate Protocol 4: Extraction of DNA form Cigarette Butts

  Materials
  • Bone specimen (usually 0.5 to 2.5 g)
  • 10% (v/v) commercial bleach (7 mM sodium hypochlorite solution)
  • 95% ethanol
  • Liquinox
  • recipeExtraction buffer (see recipe)
  • 20 mg/ml proteinase K
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • n‐butanol
  • TE buffer, pH 7.5 ( appendix 2D), sterile
  • 1% (w/ v) agarose gel in 1× TBE (unit 2.7)
  • recipe6× gel loading buffer (see recipe)
  • Sizing ladder (123 base pair DNA ladder)
  • Chisel and hammer
  • Mortar
  • Sterile aluminum oxide sanding bit
  • Rotary tool, hand‐held (e.g., Dremel)
  • Bone sanding hood
  • Blender (e.g., Waring)
  • 50‐ml conical tube
  • 15‐ml polypropylene tube
  • Rocker platform at 56°C (e.g., Nutator, Becton Dickinson Primary Care Diagnostics)
  • Centrifuge (e.g., IEC Centra MP4)
  • Centricon 100 concentrators (Amicon)
  • Polaroid 667 film
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Alternate Protocol 5: Extraction of DNA from Bones

  Materials
  • Intact tooth with roots
  • 10% (v/v) commercial bleach (7 mM sodium hypochlorite)
  • 95% and 100% ethanol
  • recipeExtraction buffer (see recipe)
  • 20 mg/ml proteinase K
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • n‐butanol
  • Sterile TE buffer, pH 7.5 ( appendix 2D)
  • 50‐ml conical tube
  • Cut‐off wheel (Dremel) or diamond cutting disk (Brasseler)
  • Dental drill and bits
  • 1.5‐ml microcentrifuge tubes
  • Rotary tool, hand‐held (e.g., Dremel)
  • 56°C incubator (or 56°C heating block)
  • Centricon 100 concentrators (Amicon)
  • Centrifuge (e.g., IEC Centra MP4)

Alternate Protocol 6: Extraction of DNA from Teeth

  • Sample envelope or stamp
  • recipe10 mg/ml proteinase K (see recipe)
  • Sterile swabs
  • 56°C water bath

Alternate Protocol 7: Chelex Extraction of DNA from Envelope Flaps and Stamps

  Materials
  • SeaKem I.D. NA agarose (FMC Bioproducts)
  • TAE buffer ( appendix 2D; also called TAN buffer)
  • 10 mg/ml ethidium bromide ( appendix 2D)
  • Yield calibrators (Lifecodes)
  • 123‐bp visual size marker (Life Technologies)
  • Recovered DNA samples for yield determination
  • recipe2× loading solution (see recipe)
  • 65°C water bath
  • Microtiter plates
  • Polaroid 667 film
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
CAUTION: Ethidium bromide is a strong carcinogen and mutagen, requiring careful handling and neutralization with bleach or filtration of the buffers to remove the chemical prior to disposal. Also see appendix 2A.NOTE: The Lifecodes Recovery Kit contains all reagents necessary to complete the protocol below.

Support Protocol 2: Yield‐Gel Determination of Quality and Quantity of Forensic DNA Samples

  Materials
  • Recovered DNA samples, MgCl 2‐free
  • TE buffer, pH 8.0 ( appendix 2D)
  • QuantiBlot Kit (Applied Biosystems) containing:
  •  Human DNA standards
  •  DNA calibrators D17Z1 biotin‐labeled probe
  •  Enzyme conjugate: HRP‐SA
  • recipeSpotting solution (see recipe)
  • Pre‐wetting solution: 0.4 M NaOH/25 mM EDTA (store in plastic bottle ≤1 year at room temperature)
  • recipeHybridization solution (see recipe)
  • recipeWash solution (see recipe)
  • 30% hydrogen peroxide (do not use beyond labeled expiration date)
  • recipeCitrate buffer (see recipe)
  • recipeChromogen solution (see recipe)
  • 3% (v/v) hydrogen peroxide (store protected from light up to 1 month at 4°C)
  • ECL detection reagents 1 and 2 (Amersham)
  • Convertible filtration manifold with 48‐well slot‐blot apparatus (Life Technologies) attached to vacuum source with pressure ≥8 to 10 in. Hg
  • Biodyne B nylon membrane (PALL)
  • Hybridization tray and lid
  • 50°C rotating water bath
  • Orbital shaker
  • MP4 Polaroid camera with type 52 black‐and‐white film or MPX one‐step Polaroid photocopier with type 1910 black‐and‐white film (for colorimetric detection)
  • Benchkote surface protectors (Whatman; for chemiluminescent detection)
  • Hyperfilm ECL or Kodak XAR X‐ray film (for chemiluminescent detection)
  • Automatic film processor (for chemiluminescent detection)
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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.
   Anderson, T.D., Ross, J.P., Lee, D.A., Roby, R.K., Canik, J.J., and Holland, M.M. 1999. A validation study for the extraction and analysis of DNA from human nail material and its application to forensic casework. J. Forensic Sci. 44:1053‐1056.
   Bisbing, R.E. 1982. The forensic identification and association of human hair. In Forensic Science Handbook (R. Saferstein, ed.) pp. 184‐221. Prentice‐Hall, Englewood Cliffs, N.J.
   Bourke, M.T., Scherczinger, C.A., Ladd, C., and Lee, H.C. 1999. NaOH treatment to neutralize inhibitors of Taq polymerase inhibitors. J. Forensic Sci. 944:1046‐1050.
   Buel, E. and Schwartz, M. 1995. The use of DAPI as a replacement for ethidium bromide in forensic DNA analysis. J. Forensic Sci. 40:275‐278.
   Collins, K.A., Rao, P. Nagesh, Hayworth, R., Schnell, S., Tap, M.P., Lantz, P.E., Geisinger, K.R., and Pettenati, M.J. 1994. Identification of sperm and non‐sperm male cells in cervicovaginal smears using fluorescence in situ hybridization: Applications in alleged sexual assault. J. Forensic Sci. 39:1347‐1355.
   Comey, C.T., Koons, B.W., Presley, K.W., Smerick, J.B., Sobieralski, C.A., Stanley, D.M., and Baechtel, F.S. 1994. DNA extraction strategy for amplified fragment length polymorphism analysis. J. Forensic Sci. 39:1254‐1269.
   DeSalle, R., Barcia, M., and Wray, C. 1993. PCR jumping in clones of 30‐million‐year‐old DNA fragments from amber preserved termites (Mastotermes electrodominicus). Experientia 49:906‐909.
   Eisenberg, A.J. 1997. Advanced DNA technologies: Automation and applications. Proc. Am. Acad. Forensic Sci. W16:7.
   Gill, P., Jeffreys, A.J., and Werrett, D.J. 1985. Forensic application of DNA “fingerprints.” Nature 318:577‐579.
   Giusti, A., Baird, M., Pasquale, S., Balazs, I., Glassberg, J. 1986. Application of deoxyribonucleic acid (DNA) polymorphisms to analysis of DNA recovered from sperm. J. Forensic Sci. 31:409‐417.
   Handt, O., Krings, M., Ward, R.H., and Paabo, S. 1996. The retrieval of ancient human DNA sequences. Am. J. Hum. Genet. 59:368‐376.
   Handt, O., Richards, M., Trommsdorff, M., Kilger, C., Simanainen, J., Georgiev, O., Bauer, K., Stone, A., Hedges, R., Schaffner, W., Utermann, G., Sykes, B., and Paabo, S. 1994. Molecular genetic analyses of the Tyrolean ice man. Science 264:1775‐1778.
   Hochmeister, M.N., Budowle, B., Jung, J., Borer, U.V., Comey, C.T., and Dirnhofer, R. 1991b. PCR‐based typing of DNA extracted from cigarette butts. Int. J. Leg. Med. 104:229‐233.
   Holland, M.M. and Parsons, T.J. 1998. Mitochondrial DNA sequence analysis–Validation and use for forensic casework. Forensic Sci. Rev. 11:21‐50.
   Holland, M.M., Roy, R., Fraser, M.D., and Liu, R.H. 1993. Application of serological and DNA methods for identification of urine specimen donors. Forensic Sci. Rev. 5:1‐14.
   Holland, M.M., Fisher, D.L., Roby, R.K., Ruderman, J., Bryson, C., and Weedn, V.W. 1995. Mitochondrial DNA sequence analysis of human remains. Crime Lab. Dig. 22:109‐115.
   Hopwood, A.J., Mannucci, A., and Sullivan, K.M. 1996. DNA typing from human faeces. Int. J. Legal Med. 108:237‐243.
   Hummel, S. and Herrmann, B. 1994. General aspects of sample preparation. In Ancient DNA (B. Herrmann and S. Hummel, eds.) pp. 59‐68. Springer‐Verlag, New York.
   Kanter, F., Baird, M., Shaler, R., and Balazs, I. 1986. Analysis of restriction fragment length polymorphisms on deoxyribonucleic acid (DNA) recovered from dried blood stains. J. Forensic Sci. 31:402‐408.
   Ladd, C., Lee, H., and Bieber, F.R. 2000. Probability of exclusion estimates in forensic analysis of complex DNA mixtures. B‐23. Abstr. American Academy of Forensic Sciences, Reno, NV.
   McNally, L., Shaler, R.C., Baird, M., Balazs, I., DeForest, P., and Kobilinsky, L. 1989a. Evaluation of deoxyribonucleic acid (DNA) isolated from human blood stains exposed to ultraviolet light, heat, humidity, and soil contamination. J. Forensic Sci. 34:1059‐1069.
   McNally, L., Shaler, R.C., Baird, M., Balazs, I., Kobilinsky, L., and DeForest, P. 1989b. Effects of environment and subtraction on deoxyribonucleic acid (DNA): The use of casework samples from New York City. J. Forensic Sci. 34:1070‐1077.
   National Research Council 1996. The evaluation of forensic DNA evidence. National Academy Press, Washington, D.C.
   Nuovo, G.J. 1994. PCR In SituHybridization. Raven Press, New York.
   Ou, C.‐Y., Moore, J.L., and Schochetman, G. 1991. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 10:442‐446.
   Paabo, S., Irwin, D.M., and Wilson, A.C. 1990. DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265:4718‐4721.
   Prince, A.M. and Andrus, L. 1992. PCR: How to kill unwanted DNA. Biotechniques 12:358‐360.
   Smith, B.C., Fisher, D.L., Weedn, V.W., Warnock, G.R., and Holland, M.M. 1993. A systematic approach to the sampling of dental DNA. J. Forensic Sci. 38:1194‐1209.
   Technical Working Group on DNA Analysis Methods (TWGDAM) 1995. Guidelines for a quality assurance program for DNA analysis. Crime Laboratory Digest 22:21‐43.
   Thomas, K. and Paabo, S. 1993. DNA sequences from old tissue remains. Methods Enzymol. 224:406‐419.
   Walsh, P.S., Metzger, D.A., and Higuchi, R. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR‐based typing from forensic material. BioTechniques 10:506‐513.
   Weir, B.S. 1996. Genetic Data Analysis II. Sinauer Associates, Sunderland, Mass.
   Weir, B.S., Triggs, C.M., Starling, L., Stonewell, L.I., Walsh, K.A.J., and Buckelton, J. 1997. Interpreting DNA mixtures. J. Forensic Sci. 42:213‐222.
   Williamson, J.M., Waye, J.S., Newall, P., Bing, D.H., and Blake, E. 1995. The use of a comprehensive approach for neutralization of PCR inhibitors found in forensic samples and its use in a homicide/sexual assault case. In Proceedings of Sixth International Symposium on Human Identification, pp. 186‐187. Promega, Madison, Wis.
   Wilson, M.R., Polanskey, D., Butler, J., DiZinno, J.A., Replogle, J., and Budowle, B. 1995. Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18:662‐669.
   Word, C.J., Sawosik, T., and Bing, D.H. 1997. Summary of validation studies from twenty‐six forensic laboratories in the United States and Canada on the use of the AmpliType PM PCR Amplification and Typing Kit. J. Forensic Sci. 42:39‐48.
Key References
   Gill et al., 1985. See above.
  Describes the differential extraction procedure used for sexual assault samples.
   TWGDAM, 1995. See above.
  Details guidelines for a quality assurance program for forensic DNA testing; these guidelines became the interim rules for DNA forensic testing under the Omnibus Crime Bill passed October, 1994, which included legislation on forensic DNA testing; they form the basis for the rules formulated by the DNA Advisory Board mandated in that same piece of legislation to formulate rules for forensic DNA testing in the United States.
   Walsh et al., 1991. See above.
  Describes the Chelex method for isolation of DNA from forensic samples.
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