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An easily automated, closed-tube forensic DNA extraction procedure using a thermostable proteinase

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Abstract

Most standard procedures for extracting DNA from forensic substrates involve manipulations that expose the sample to potential contamination and which reduce yields. Furthermore, most methods require centrifugation and/or solvent extraction steps that render them difficult to automate. We describe a simple closed-tube DNA extraction procedure using a proteinase from the thermophilic Bacillus species EA1 that produces good DNA yields from a wide range of forensic substrates. The reaction is controlled by a temperature shift regime programmed into a thermal cycler and so eliminates the need for solvent extraction or column purification. The new method is ideally suited to forensic samples where exposure to extraneous contaminating DNA must be avoided. In addition, The simplicity of the procedure makes it suitable for automation.

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References

  1. McHale R, Stapleton PM, Bergquist PL (1991) A rapid method for the preparation of samples for PCR. Biotechniques 10:20–22

    CAS  PubMed  Google Scholar 

  2. Borges KM, Bergquist PL (1993) Pulsed-field gel electrophoresis study of the genome of Caldocellum saccharolyticum. Curr Microbiol 27:15–19

    CAS  Google Scholar 

  3. Collet C (2001) Proteases suitable for use in molecular biology. Master of philosophy thesis, University of Waikato, Hamilton, NZ

  4. Coolbear T, Eames CW, Casey Y, Daniel RM, Morgan HW (1991) Screening of strains identified as extremely thermophilic bacilli for extracellular proteolytic activity and general properties of the proteinases from two of the isolates. J Appl Bacteriol 71:252–264

    Google Scholar 

  5. Coolbear T, Whittaker JM, Daniel RM, Morgan HW (1992) The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus strain EA1. Biochem J 286:367–374

    Google Scholar 

  6. Saul DJ, Williams LC, Toogood HS, Daniel RM, Bergquist PL (1996) Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation. Biochim Biophys Acta 1308:74–80

    Article  CAS  PubMed  Google Scholar 

  7. Linch C, Smith SL, Prahlow JA (1998) Evaluation of the human hair root for DNA typing subsequent to microscopic comparison. J Forensic Sci 43:305–314

    CAS  PubMed  Google Scholar 

  8. Sweet D, Lorente M, Lorente J, Valenzuela A, Villanueva E (1997) An improved method to recover saliva from human skin: the double swab technique. J Forensic Sci 42:320–322

    CAS  PubMed  Google Scholar 

  9. Walsh PS, Metzge D, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10:506–513

    CAS  PubMed  Google Scholar 

  10. Sambrook J, Fritsch DW (2001) Molecular cloning, a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, USA

  11. Moretti T, Baumstark AL, Defenbaugh BS, Keys KM, Smerick JB, 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 3:647–660

    Google Scholar 

  12. Gill P, Sparkes RL, Pinchin R, Clayton T, Whitaker JP, Buckleton J (1998) Interpreting simple STR mixtures using allelic peak areas. Forensic Sci Int 91:41–53

    Article  CAS  PubMed  Google Scholar 

  13. Clayton T, Whitaker JP, Sparkes RL, Gill P (1998) Analysis and interpretation of mixed forensic stains using DNA STR profiling. Forensic Sci Int 91:55–70

    Article  CAS  PubMed  Google Scholar 

  14. Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K (1994) Identification of the heme compound co-purified with deoxyribonucleic acid (DNA) from bloodstains. A major inhibitor of the polymerase chain reaction (PCR) amplification. J Forensic Sci 39:362–372

    CAS  PubMed  Google Scholar 

  15. Klintschar M, Neuhuber F (2000) Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis. J Forensic Sci 45:669–673

    CAS  PubMed  Google Scholar 

  16. Matsuda H, Imaizumi K, Kubota S, Miyasaka S, Yoshino M, Seta S (1997) Technical investigation of DNA extraction from a single hair shaft. Rep Nat Res Inst Police Sci 50:23–28

    CAS  Google Scholar 

  17. Van Oorschot RAH, Phelan DG, Furlong S, Scarfo GM, Holding NL, Cummins MJ (2003) Are you collecting all the available DNA from touched objects? In: Brinkmann B, Carracedo A (eds) Progress in forensic genetics 9. Elsevier, Amsterdam, pp 803–807

  18. Jehaes E, Toprak K, Vanderheyen H, Pfeiffer H, Brinkmann B, Decorte R (2001) Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic analysis: the historical case of the putative heart of Louis XVII. Int J Legal Med 115:135–141

    CAS  PubMed  Google Scholar 

  19. Hellmann A, Rohleder U, Schmitter H, Wittig M (2001) STR typing of human telogen hairs—a new approach. Int J Legal Med 114:269–273

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are indebted to Professor Roy Daniel of the University of Waikato, Hamilton, New Zealand for advice and for providing the EA1 proteinase used in this work.

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Correspondence to D. J. Saul.

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Moss, D., Harbison, SA. & Saul, D.J. An easily automated, closed-tube forensic DNA extraction procedure using a thermostable proteinase. Int J Legal Med 117, 340–349 (2003). https://doi.org/10.1007/s00414-003-0400-9

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  • DOI: https://doi.org/10.1007/s00414-003-0400-9

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