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

Abstract

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.

Comparison of Two Capillary Gel Electrophoresis Systems for Clostridium difficile Ribotyping, Using a Panel of Ribotype 027 Isolates and Whole-Genome Sequences as a Reference Standard

“…QIAxcel is an automated electrophoresis platform that can deal with up to 96 samples per run with high efficiency and a turnaround time of 0.2 to 1.5 h depending on the number of specimens. As the method does not require fluorescein-labeled primers, it is simple to per- form and potentially suitable for a clinical laboratory (4,5,9). The results can be exported in either electropherogram (similar to the SCGE result shown in Fig.…”

Evaluation of Degradation in DNA from Males with a Quantitative Gender Typing, Endpoint PCR Multiplex

“…As expected, qPCR products from large template decreased faster in degraded DNA than did product from small template; however, Hughes‐Stamm et al. found no statistically significant correlation between the degradation predicted by the assay and actual sample quality. This result is in contrast to our findings with Q‐TAT, which did show a strong correlation between predicted and actual template integrity as assessed by STR typing.…”

Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR

“…These findings demonstrate that the distribution of degradation ratio and number of detectable loci varies depending on the method of degradation; therefore, it is necessary to examine the miscellaneous DNA damage to predict the number of detectable loci by measuring the degradation ratio. Recently, comparative studies for typing of degraded DNA using STR, Mini-STR, or SNP analysis were reported [22,23]. With a combination of other typing approaches (e.g., Mini-STR or SNP analysis), prediction of the number of detectable loci based on the degradation ratio could help to select an appropriate approach (e.g., STR, Mini-STR, or SNP analysis) for obtaining an informative DNA profile, and lead to effective utilization of a limited amount of DNA extracted from casework samples.…”