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DEGRADED DNA

12 Apr

DEGRADED DNA

Figure 7.9 A mixture of two individuals will lead to up to four peaks at each locus. The area between the dotted lines represents the zone where the minor component of the mixture can be interpreted. The lower dotted line represents 15% and 60% of the major peaks – below 15% is a zone where stutter peaks from the major alleles can occur and peaks, below 60% cannot be easily explained by peak imbalance. At this locus the major component can be interpreted as 13-15 while the minor component’s genotype is 16 – 20

Degraded DNA
Many samples that are collected from a crime scene may have been exposed to the environment for hours, days, or even longer if the crime scene has gone undetected. When DNA analysis is being used to identify human remains, the remains may be several years old before they are analysed or may have been exposed to severe envi- ronmental insult such as high temperatures. In all these circumstances the DNA in the

ASSESSMENT OF STR PROFILES

Figure 7.10 The profile was generated using the AmpFlSTR® Profiler Plus® kit from Applied Biosys- tems. The DNA was extracted from a bone recovered from a Scottish loch after approximately 30 years. The profile is typical of a degraded profile with a gradual reduction in the amount of product as the amplicons increase in size (see plate section for full-colour version of this figure) cellular material will not be in pristine condition and will have degraded. This leads to a characteristic DNA profile with over amplification of the smaller loci and the suc- cessful amplification declines with the size of the alleles. Figures 7.10 and 7.11 show two examples of degraded DNA sample; the first one is from a bone sample that had been in water for 30 years. The small loci have over amplified whereas the larger loci are barely detectable [22] – the decrease in amplification is gradual as the length of the alleles increases. In the second example an example of locus drop out can be seen, the first two blue loci, D3S1358 and vWA have amplified successfully but there is no FGA allele. This

Figure 7.11 The profile was generated using the AmpFlSTR BlueTM kit from Applied Biosystems. The DNA was extracted from muscle tissue recovered from a plane crash. The muscle had been subjected to high temperatures and the DNA was highly degraded – no amplification products were detected from the FGA locus. The size standard is also shown in by non-shaded peaks (see plate section for full-colour version of this figure)

REFERENCES
profile is from human muscle tissue that had been exposed to high temperatures and has degraded to the extent that there is very little or no DNA that is 200 bp or longer [23]. The interpretation of degraded profiles can be difficult and particular attention has to be taken when homozygous loci are detected – are they really homozygous and not heterozygous with one of the alleles having dropped out? When the levels are very low, if there is enough material the PCR is carried out in duplicate, as with LCN PCR, to minimize the possibility of generating an incorrect profile. To assist with the analysis of degraded DNA, a series of multiplexes have been developed with the primers positioned close to the core repeats of the STRs, thereby minimizing the lengths of the amplicons [24-28].

References

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3.Schlotterer, C., and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Research 20, 211-215.

4.Gill, P., et al. (1997) Development of guidelines to designate alleles using an STR multiplex system. Forensic Science International 89, 185-197.

5.Corporation, T.P.-E. (1999) AmpFISTR SGM PlusTM PCR Amplification Kit – User’s Manual.

6.Clark, J.M. (1988) Novel non-templated nucleotide addition-reactions catalyzed by procaryotic and eukaryotic DNA-polymerases. Nucleic Acids Research 16, 9677-9686.

7.Bohnert, M., et al. (2001) Transfer of biological traces in cases of hanging and ligature strangu- lation. Forensic Science International 116, 107-115.

8.Esslinger, K.L., et al. (2004) Using STR analysis to detect human DNA from exploded pipe bomb devices. Journal of Forensic Sciences 49, 481-484.

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10.Wiegand, P., and Kleiber, M. (1997) DNA typing of epithelial cells after strangulation. Interna- tional Journal of Legal Medicine 110, 181-183.

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

12.Gill, P., et al. (2000) An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Science International 112, 17-40.

13.Kloosterman, A.D., and Kersbergen, P. (2003) Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci. International Congress Series 1239, 795-798.

14.Whitaker, J.P., et al. (2001) A comparison of the characteristics of profiles produced with the AMPFlSTR® SGM PlusTM multiplex system for both standard and low copy number (LCN) STR DNA analysis. Forensic Science International 123, 215-223.

15.Gill, P., et al. (2006) DNA commission of the International Society of Forensic Genetics: recom- mendations on the interpretation of mixtures. Forensic Science International 160, 90-101.

16.Clayton, T.M., et al. (1998) Analysis and interpretation of mixed forensic stains using DNA STR profiling. Forensic Science International 91, 55-70.

17.Lygo, J.E., et al. (1994) The validation of short tandem repeat (Str) loci for use in forensic casework. International Journal of Legal Medicine 107, 77-89.

18.Sparkes, R., et al. (1996) The validation of a 7-locus multiplex STR test for use in forensic casework.1. Mixtures, ageing, degradation and species studies. International Journal of Legal Medicine 109, 186-194.

19. Evett, I.W., et al. (1998) Taking account of peak areas when interpreting mixed DNA profiles. Journal of Forensic Sciences 43, 62-69.

20. Gill, P., et al. (1998) Interpreting simple STR mixtures using allele peak areas. Forensic Science International 91, 41-53.

21. Bill, M., et al. (2005) PENDULUM-a guideline-based approach to the interpretation of STR mixtures. Forensic Science International 148, 181-189.

22. Goodwin, W., et al. (2003) The identification of a US serviceman recovered from the Holy Loch, Scotland. Science and Justice 43, 45-47.

23. Goodwin, W., et al. (1999) The use of mitochondrial DNA and short tandem repeat typing in the identification of air crash victims. Electrophoresis 20, 1707-1711.

24. Dixon, L.A., et al. (2006) Analysis of artificially degraded DNA using STRs and SNPs – results of a collaborative European (EDNAP) exercise. Forensic Science International 164, 33-44.

25. Gill, P., et al. (2006) The evolution of DNA databases – recommendations for new European STR loci. Forensic Science International 156, 242-244.

26. Coble, M.D., and Butler, J.M. (2005) Characterization of new MiniSTR loci to aid analysis of degraded DNA. Journal of Forensic Sciences 50, 43-53.

27. Drabek, J., et al. (2004) Concordance study between Miniplex assays and a commercial STR typing kit. Journal of Forensic Sciences 49, 859-860.

28. Butler, J.M., et al. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. Journal of Forensic Sciences 48, 1054-1064.

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