The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains*

The University of Tennessee Medical Center at Knoxville, Knoxville, Tennessee, United States
Journal of Forensic Sciences (Impact Factor: 1.16). 04/2006; 51(2):351-6. DOI: 10.1111/j.1556-4029.2006.00077.x
Source: PubMed


A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.

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    • "Biochemical studies have increased in frequency during the past several years. For example, researchers have used decomposing remains to investigate methods of recovering degraded DNA from bone, hair, fingernails, and fingerprints and to test the effects of different environmental conditions on the quality and quantity of DNA recovery (i.e., open ground, shaded ground, burial, and water) (Opel et al., 2006). Vass et al. (1992, 2002, 2004, 2008) conducted seminal research in estimating time since death from the amounts of volatile fatty acids and odor compounds. "

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    • "Generally, the certainty of identity with one locus is less than 0.01, and is 1.0×10−15 for 12 or more loci, making STR a very effective method for sample identification [2], [3]. Despite its efficacy, improvements to STR assays have been required for forensic analysis on highly-degraded DNA samples, namely reducing amplicon size to increase amplification efficiency [4], [5]. Previous studies on degraded DNA have demonstrated that STR analysis using amplicons of 280 nt or less generates 48% more genotype profiles than would be possible with longer STRs [5]. "
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    ABSTRACT: Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.
    PLoS ONE 02/2014; 9(2):e88163. DOI:10.1371/journal.pone.0088163 · 3.23 Impact Factor
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    • "For example, the focus has been placed on mini-STRs with amplicon sizes between 70-280 bp, based on the principle that smaller fragments can often still be amplified when DNA is already highly degraded (16-18). Nowadays, a wide range of commercially available STR-kits and self-made PCRs, especially developed for highly degraded samples is available (19-22). However, there is a considerable difference in efficiency, costs, and the processing time for each assay. "
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    ABSTRACT: Aim To comparatively test nine commercially available short tandem repeat (STR)-multiplex kits (PowerPlex 16, 16HS, ES, ESI17, ESX17, S5 [all Promega]; AmpFiSTR Identifiler, NGM and SEfiler [all Applied Biosystems]) for their efficiency and applicability to analyze ancient and thus highly degraded DNA samples. Methods Fifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocols. Data were systematically compared to find the most meaningful and sensitive assay. Results The ESI, ESX, and NGM kits showed the best overall results regarding amplification success, detection rate, identification of heterozygous alleles, sex determination, and reproducibility of the obtained data. Conclusion Since application of these three kits enables the employment of different primer sequences for all the investigated amplicons, a combined application is recommended for best possible and – most importantly – reliable genetic analysis of ancient skeletal material or otherwise highly degraded samples, eg, from forensic cases.
    Croatian Medical Journal 10/2012; 53(5):416-22. DOI:10.3325/cmj.2012.53.416 · 1.31 Impact Factor
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