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Assessment of DNA degradation and the genotyping success of highly degraded samples

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DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples. We report a comparative study of genotyping using STRs, mini-STRs and single nucleotide polymorphisms (SNPs) with template at different levels of degradation in varying amounts. Two methods of assessing quantity and quality of a DNA sample prior to genotyping were investigated. The QIAxcel capillary gel electrophoresis system provided a rapid, cost effective screening method for assessing sample quality. A real-time quantitative PCR (qPCR) assay was able to simultaneously quantify total human DNA, male DNA, DNA degradation and PCR inhibition. The extent of DNA degradation could be assessed with reasonable accuracy to 62.5 pg and genomic targets could be quantified to a lower limit of 15.6 pg. The qPCR assay was able to detect male DNA to a lower limit of 20 pg in a 1:1,000 background of female DNA. By considering the amount of DNA and the degradation ratio of a sample, a general prediction of genotyping success using AmpFlSTR® Profiler Plus®, MiniFiler™ kits and SNP analysis can be made. The results indicate mini-STRs and SNP markers are usually more successful in typing degraded samples and in cases of extreme DNA degradation (≤200 bp) and template amounts below 250 pg, mini-STR and SNP analysis yielded significantly more complete profiles and lower match probabilities than corresponding STR profiles.

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We would like to thank the Australian and New Zealand Forensic Science Society (ANZFSS) for funding travel to the 6th International Society of Applied Biological Sciences (ISABS) Conference, 2009 where a portion of this data was presented as a poster.

Any Australian ethical permission necessary for this project has been granted and ethical standards have been adhered to.

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The authors declare that they have no conflict of interest.

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Correspondence to Angela van Daal.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Online Resource 1

Amplicon length, fluorescence labels, primer and probe concentrations of each target sequence included in the quadruplex qPCR assay. (PDF 22 kb)

Online Resource 2

The RS number and amplicon length of the 53 SNPs genotyped (PDF 22 kb)

Online Resource 3

Sensitivity data of the quadruplex qPRC assay and degradation ratios with undegraded and degraded samples. (PDF 22 kb)

Online Resource 4

Quantification results of male and female mixed samples indicating male: female ratios generated by the quadruplex assay. (PDF 21 kb)

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R. Hughes-Stamm, S., J. Ashton, K. & van Daal, A. Assessment of DNA degradation and the genotyping success of highly degraded samples. Int J Legal Med 125, 341–348 (2011).

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