A quantitative PCR assay for the assessment of DNA degradation in forensic samples

Swango, Katie L.; Timken, Mark D.; Chong, Mavis Date; Buoncristiani, Martin R.

doi:10.1016/j.forsciint.2005.04.034

Cited by 98 publications

(74 citation statements)

References 16 publications

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“…During PCR 2, the IACs fragments are amplified with a common ROX-labelled ( ) forward primer, allowing the detection of the markers with the human DNA profile and internal size standard. [24] and Asamura et al [25]; after profiling with SGM Plus s (without IACs), the 10 min time point was selected for testing with the IACs because it produced a partial profile, with amplification of the small loci and dropout/reduced signal of the larger loci. In every case, controls containing human DNA only and IACs only were run with the samples.…”

Section: Amplification Of Iacs With Human Dnamentioning

confidence: 99%

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

et al. 2011

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DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

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Section: Amplification Of Iacs With Human Dnamentioning

confidence: 99%

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

et al. 2011

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“…An artificially degraded DNA sample series was prepared as described earlier [13,26]: 11.2 μg of male genome DNA was mixed with 10× DNase I reaction buffer (Invitrogen, Carlsbad, CA, USA) and sterile water at a total volume of 110 μl. Of the reaction mixture, 10 μl was removed as control DNA undigested by DNase, and 2.5 U of DNase I (Invitrogen) was added to the remaining 100 μl reaction mixture.…”

Section: Analysis Of Degraded Dnamentioning

confidence: 99%

Evaluation of miniY-STR multiplex PCR systems for extended 16 Y-STR loci

et al. 2007

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We developed three short amplicon Y-chromosomal short tandem repeat (miniY-STR) polymerase chain reaction multiplex systems for 16 Y-STR loci (DYS441, DYS446, DYS462, DYS481, DYS485, DYS495, DYS505, DYS510, DYS511, DYS549, DYS 575, DYS578, DYS593, DYS618, DYS638, and DYS643), using newly designed primer sets. In an assay of 238 Japanese males using the three miniY-STR systems, amplification product lengths ranged from 91 to 151 bp for all 16 Y-STR loci. We identified 212 different haplotypes among the 238 individuals, finding haplotype diversity and discrimination capacity of 0.9974 and 0.8908, respectively. An assay of degraded DNA samples using the three miniY-STR multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. In conclusion, analyses of miniY-STR multiplex systems will play an important role in forensic applications involving degraded DNA samples for which genotyping using only commercial kits is ill-suited.

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“…Multiplex real-time quantitative PCR (qPCR) assays which simultaneously quantify total human genomic DNA, male DNA, the extent of DNA degradation and the presence of PCR inhibitors have been previously described [17,18]. Further to Hudlow et al, 2008 [17] we report a capillary gel electrophoresis method of assessing DNA degradation in addition to a qPCR assay.…”

Section: Introductionmentioning

confidence: 99%

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

2010

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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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A quantitative PCR assay for the assessment of DNA degradation in forensic samples

Cited by 98 publications

References 16 publications

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Evaluation of miniY-STR multiplex PCR systems for extended 16 Y-STR loci

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

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A quantitative PCR assay for the assessment of DNA degradation in forensic samples

Cited by 98 publications

References 16 publications

Development of internal amplification controls for DNA profiling with the AmpFℓSTR® SGM Plus® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR® SGM Plus® kit

Evaluation of miniY-STR multiplex PCR systems for extended 16 Y-STR loci

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

Contact Info

Product

Resources

About

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit