A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: A diagnostic tool for STR typing

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

doi:10.1016/j.fsigen.2007.09.001

Cited by 80 publications

(48 citation statements)

References 24 publications

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“…Further to Hudlow et al, 2008 [17] we report a capillary gel electrophoresis method of assessing DNA degradation in addition to a qPCR assay. We also investigate the comparative genotyping success of highly degraded samples using STR, mini-STR and SNP typing systems.…”

Section: Introductionmentioning

confidence: 89%

“…qPCR Degradation assessment utilised a modified quadruplex qPCR assay previously described [17]. The four targets included two autosomal (TH01 and CSF), one malespecific target (SRY) and a synthetic oligonucleotide internal PCR control (IPC).…”

Section: Methodsmentioning

confidence: 99%

“…Modifications to the previously published assay include five-prime dyes and primer concentrations (Online Resource 1). Primer and probe sequences were unchanged as in Hudlow [17]. The two-step qPCR protocol consisted of an initial 15 min 95°C polymerase activation step, followed by 40 cycles of 60 s of denaturation (94°C) and 90 s of combined annealing/extension (60°C).…”

Section: Methodsmentioning

confidence: 99%

“…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: 94%

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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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Section: Introductionmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

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

2010

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“…DNA was extracted from cat, pig, horse, cow, and chicken whole blood and quantified by an electrophoretic method in a 1% agarose gel containing ethidium bromide. Other previously quantified genomic DNA samples provided by the Jan Bashinski laboratory included: fish, monkey, rat (Zyagen Laboratories, San Diego, CA, USA), mouse (Promega, Madison, WI, USA), Bacillus subtilis, Staphylococcus epidermidis, Candida albicans (ATCC, Manassas, VA, USA), Escherichia coli, and Clostridium perfringens (Sigma, St. Louis, MO, USA) (19). A human buccal sample was extracted using the Epicenter QuickExtract protocol (Epicenter).…”

Section: Methodsmentioning

confidence: 99%

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

Dayton

Koskinen²,

Tom

et al. 2009

Croat Med J

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Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker.Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra-and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. ResultsThe kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. ConclusionThe kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study's evaluation to comply with the quality standards expected for forensic casework.

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CE analysis and molecular characterisation of depurinated DNA samples

Fattorini¹,

Marrubini²,

Sorçaburu-Cigliero³

et al. 2011

Electrophoresis

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A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6) s(-1) for adenine and 6.27×10(-6) s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing.

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A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: A diagnostic tool for STR typing

Cited by 80 publications

References 24 publications

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

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

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

CE analysis and molecular characterisation of depurinated DNA samples

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