12 Apr


Figure 5.5 The PCR laboratory is designed so that the work flows through the different processes in one direction starting with sample reception and forensic biology and finishing with the post-PCR analysis. The samples are passed through air-lock hatches to minimize the possibility of any material being transferred from post-PCR to pre-PCR areas. Access to the pre- and post-PCR laboratories is through different changing areas and dedicated staff will work in either pre- or post-PCR areas. Positive air pressure in pre-PCR areas and negative pressure in post-PCR rooms also reduces the possibility of introducing any contamination into the pre-PCR areas efficiently. The PCR set-up introduces another positive and negative control: the pos- itive control involves setting-up a PCR with DNA of known origin and whose profile is known. Successful analysis demonstrates that the reaction worked. In the negative control PCR, water replaces the DNA to monitor for contamination in the reagents or introduced during the PCR set up.


The most potent source of contamination is previously amplified PCR products. Fol- lowing a PCR there are millions of copies of the target sequence that can potentially contaminatesubsequentreactions.EachtimeaPCRtubeisopenedthereissomeaerosol spray and a single droplet of aerosol will contain thousands of copies of the amplified target, resulting in transfer of some of the amplified product. The fundamental feature of any laboratory that engages in PCR analysis is that there must be physical separation of the pre-PCR and the post-PCR analysis to minimize the possibility of contaminating DNA extractions and PCR set-ups with amplified material. In addition to the two phys- ical spaces there should also be dedicated equipment, protective clothing and reagents for each area. There must be a unidirectional work flow through the laboratory – PCR products must never be brought back into the pre-PCR part of the laboratory. There must also be temporal separation of tasks – it is not possible for a scientist who has been working in the post-PCR to then work in the pre-PCR area without the possibility of introducing contamination; an overnight break before returning to the pre-PCR area is normally recommended. Larger laboratories will have scientists who are dedicated to only the pre- or the post-PCR analysis.

Further reading

Dieffenbach C.W., and Dveksler G.S. (2003) PCR Primer: A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press.

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2.Mullis, K.B., and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase- atalyzed chain-reaction. Methods in Enzymology 155, 335-350.

3.Saiki, R.K., et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restric- tion site analysis for diagnosis of sickle-cell anemia. Science 230, 1350-1354.

4.Li,H.H.,etal.(1990)DirectelectrophoreticdetectionoftheallelicstateofsingleDNA-molecules in human sperm by using the polymerase chain-reaction. Proceedings of the National Academy of Sciences of the United States of America 87, 4580-4584.

5.Li, H.H., et al. (1988) Amplification and analysis of DNA-sequences in single human-sperm and diploid-cells. Nature 335, 414-417.

6.Saiki, R.K., et al. (1986) Analysis of enzymatically amplified beta-globin and HLA-DQ-α DNA with allele-specific oligonucleotide probes. Nature 324, 163-166.

7.Stoneking, M., et al. (1991) Population variation of human MtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes. American Journal of Human Genetics 48, 370-382.

8.Blake,E.,etal.(1992)Polymerasechain-reaction(PCR)amplificationandhuman-leukocyteanti- gen (HLA)-DQ-α oligonucleotide typing on biological evidence samples – casework experience. Journal of Forensic Sciences 37, 700-726.

9.Jeffreys, A.J., et al. (1988) Amplification of human minisatellites by the polymerase chain- reaction Towards DNA Fingerprintin gofsinglecells. Nucleic Acids Research 16,10953-10971.

10.Boerwinkle, E., et al. (1989) Rapid typing of tandemly repeated hypervariable loci by the poly-merase chain-reaction-application to the apolipoprotein-B 3′ hypervariable region. Proceedings of the National Academy of Sciences of the United States of America 86, 212-216.

11.Budowle, B., et al. (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high- resolution page. American Journal of Human Genetics 48, 137-144.

12.Horn,G.T.,etal.(1989)AmplificationofahighlypolymorphicVNTRsegmentbythepolymerase chain-reaction. Nucleic Acids Research 17, 2140-2140.

13.Kasai, K., et al. (1990) Amplification of a variable number of tandem repeats (VNTR) locus (PMCT118) by the polymerase chain-reaction (PCR) and its application to forensic-science. Journal of Forensic Sciences 35, 1196-1200.

14.Rand, S., et al. (1992) Population-genetics and forensic efficiency data of 4 AmpFLPs. Interna- tional Journal of Legal Medicine 104, 329-333.

15.Sajantila, A., et al. (1991) The polymerase chain-reaction and postmortem forensic identity testing-application of amplified D1S80 and HLA-DQ-α loci to the identification of fire victims. Forensic Science International 51, 23-34.

16.Edwards, A., et al. (1991) DNA typing and genetic-mapping with trimeric and tetrameric tandem repeats. American Journal of Human Genetics 49, 746-756.

17.Hagelberg, E., et al. (1991) Identification of the skeletal remains of a murder victim by DNA analysis. Nature 352, 427-429.

18.Chien, A., et al. (1976) Deoxyribonucleic-acid polymerase from extreme thermophile thermus aquaticus. Journal of Bacteriology 127, 1550-1557.

19.Saiki, R.K., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA-polymerase. Science 239, 487-491.

20.Daquila, R.T., et al. (1991) Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acids Research 19, 3749-3749.

21.Birch, D.E., et al. (1996) Simplified hot start PCR. Nature 381, 445-446.

22.Moretti, T., et al. (1998) Enhancement of PCR amplification yield and specificity using AmpliTaq Gold (TM) DNA polymerase. Biotechniques 25, 716-722.

23.Budowle, B., et al. (2001) STR primer concordance study. Forensic Science International 124, 47-54.

24.Rychlik,W.,andRhoads,R.E.(1989)Acomputer-programforchoosingoptimaloligonucleotides for filter hybridization, sequencing and in-vitro Amplification of DNA. Nucleic Acids Research 17,8543-8551.

25.Rozen, S., and Skaletsky, H.J. (2000) Primer3 on the WWW for general users and for biolo- gist programmers.. In Bioinformatics Methods and Protocols: Methods in Molecular Biology (Krawetz, S., and Misener, S., eds), Humana Press, pp. 365-386

26.Takagi, M., et al. (1997) Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Applied and Environmental Microbiology 63, 4504-4510

27.Applied Biosystem. A feature guide for PCR enzymes. Avaliable at

28.Gill, P. (2001) Application of low copy number DNA profiling. Croatian Medical Journal 42, 229-232.

29.Wilson, I.G. (1997) Inhibition and facilitation of nucleic acid amplification. Applied and Envi-ronmental Microbiology 63, 3741-3751.

30.Akane, A., et al. (1994) Identification of the heme compound copurified with deoxyribonucleic-acid(DNA)frombloodstains,amajorinhibitorofpolymerasechain-reaction(PCR)amplification. Journal of Forensic Sciences 39, 362-372.

31.Akane, A., et al. (1993) Purification of forensic specimens for the polymerase chain-reaction (PCR) analysis. Journal of Forensic Sciences 38, 691-701.

32.Defranchis, R., et al. (1988) A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Research 16, 10355-10355.

33.Lantz, P.G., et al. (1997) Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. Journal of Microbiological Methods 28, 159-167.

34.Monteiro, L., et al. (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. Journal of Clinical Microbiology 35, 995-998.

35.Tsai, Y.L., and Olson, B.H. (1992) Detection of low numbers of bacterial-cells in soils and sediments by polymerase chain-reaction. Applied and Environmental Microbiology 58, 754-757.

36.Khan, G., et al. (1991) Inhibitory effects of urine on the polymerase chain-reaction for cy-tomegalovirus DNA. Journal of Clinical Pathology 44, 360-365.

37.Abu Al-Soud, W., and Radstrom, P. (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Applied and Environ-mental Microbiology 64, 3748-3753.

38. Kreader, C.A. (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Applied and Environmental Microbiology 62, 1102-1106.

39. Larkin, A., and Harbison, S.A. (1999) An improved method for STR analysis of bloodstained denim. International Journal of Legal Medicine 112, 388-390.

40. Kontanis, E.J., and Reed, F.A. (2006) Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors. Journal of Forensic Sciences 51, 795-804.

41. Rutty, G.N., et al. (2003) The effectiveness of protective clothing in the reduction of potential DNA contamination of the sceneofcrime.International Journal of Legal Medicine117,170-174.

42. Port, N.J., et al. (2006) How long does it take a static speaking individual to contaminate the immediate environment. Forensic Science Medicine and Pathology 2, 157-164.

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