Challenging DNA: Assessment of a range of genotyping approaches for highly degraded forensic samples

Fondevila, M.; Phillips, Christopher; Naverán, Nuria; Cerezo, María; Rodríguez, Amelia; Calvo, R. Castro; Fernández, Luis Moreno; Carracedo, Ángel; Lareu, Marı́a Victoria

doi:10.1016/j.fsigss.2007.10.057

Cited by 45 publications

(33 citation statements)

References 5 publications

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“…Mini-STRs and SNPs showed considerable increases in loci retrieval across all input amounts <1 ng compared to STRs. These results support previous studies demonstrating the enhanced performance and sensitivity of the AmpFlSTR® MiniFiler™ amplification kit [20,21] and SNP genotyping [2,13,15,22] for typing degraded samples. This may be attributed to reduced amplicon size, improved kit chemistry, genotyping platforms or a combination of all factors.…”

Section: Mixturessupporting

confidence: 91%

“…DNA typing using industry standard short tandem repeats (STRs) often becomes the principle means of identification [1]. DNA becomes progressively more fragmented as biological tissue degrades and this results in a decreasing ability to gain a complete STR profile [2]. The successful genotyping of samples exhibiting very high levels of DNA degradation can be further complicated by the availability of only minute quantities of material.…”

Section: Introductionmentioning

confidence: 99%

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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: Mixturessupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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

2010

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“…First, the amplicon sizes of STR-based markers range from approximately 100-400 bp. In highly degraded samples, target amplicons are usually less than 200 bp in length [9][10][11]; thus, some routinely configured loci will not be typeable. Second, artifacts, such as stutter peaks, can add ambiguity to mixture analysis.…”

Section: Introductionmentioning

confidence: 99%

A validation study of the Qiagen Investigator DIPplex® kit; an INDEL-based assay for human identification

LaRue

King

et al. 2012

Int J Legal Med

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Marker sets that are based on small insertion/deletion (INDEL) alleles can serve as useful supplementary or stand-alone assays for human identification. A validation study has been performed on a human identification assay based on a panel of 30 INDELs and amelogenin using the Investigator DIPplex® kit (Qiagen). The assay was able to type DNA from a number of forensically relevant sample types and obtain full profiles with 62 pg of template DNA and partial profiles with as little as 16 pg of template DNA. The assay is reproducible, precise, and non-overlapping alleles from minor contributors were detectable in mixture analysis ranging from 6:1 to 19:1 mixtures. Population studies were performed on the 30 indels, and there were no significant departures from Hardy-Weinberg equilibrium or significant linkage disequilibrium between the markers (after correction for sampling). In all populations, the random match probability was 1.43 × 10(-11) or less, and the power of exclusion was greater than .999999999. We also discovered several microvariant alleles in our population samples. The data support that the Investigator DIPplex® kit provides a powerful supplement or stand-alone capability for human identity testing.

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“…However, as the number of applications for DNA technology expands, inevitable backlogs have occurred due to increasing sample loads. Factors such as time lapse between the incident and sample recovery, exposure to external elements, and storage conditions may all result in sample degradation [4][5][6]. Hence, it is critical to be able to efficiently recover and reliably analyze the evidence collected.…”

Section: Introductionmentioning

confidence: 99%

The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs

Nori

McCord

2015

Anal Bioanal Chem

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This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

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Challenging DNA: Assessment of a range of genotyping approaches for highly degraded forensic samples

Cited by 45 publications

References 5 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

A validation study of the Qiagen Investigator DIPplex® kit; an INDEL-based assay for human identification

The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs

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