Dataset Information

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

ABSTRACT:

Aim

To comparatively test nine co mmercially 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, e.g., from forensic cases.

SUBMITTER: Harder M

PROVIDER: S-EPMC3494155 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

Harder Melanie M Renneberg Rebecca R Meyer Patrick P Krause-Kyora Ben B von Wurmb-Schwark Nicole N

Croatian medical journal 20121001 5

<h4>Aim</h4>To comparatively test nine co mmercially 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.<h4>Methods</h4>Fifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocol ...[more]

PMID: 23100203

Similar Datasets

Project description:Due to its proficiency to provide the most discriminating results for forensic applications, medical research and anthropological studies, multiplex PCR based STR analysis has been established as the most efficient technique in the forensic DNA analysis. Several multiplex amplification kits based on 4, 5 and 6 dyes chemistry are commercially available and used in forensic DNA typing across the globe. These multiplex PCR systems are routinely used for amplification of multiple STR loci (Autosomal, Y and/or X STR's) in the DNA extracted from various biological samples. In the routine forensic DNA testing, DNA profile obtained is compared with the DNA profile of the reference sample, which takes a certain turnaround time and employs costly lab resources. Successive development in forensic DNA typing have resulted in advent of improved multiplex kits which have reduced the effective analysis time, cost and minimized the number of steps required in comparison to conventional forensic DNA typing. Specialized direct amplification compatible multiplex kits are also available nowadays. These kits are relatively costlier but still require few pre-processing steps, which does not make them worth the hefty cost. Herein, this study, we have used non-direct multiplex STR kits to assess their efficacy for direct amplification. In the present study, 103 saliva samples were directly amplified without any pre-treatment of the samples using thirteen non-direct multiplex kits (4 dyes, 5 dyes and 6 dyes chemistry based) for forensic DNA typing. Here, we report a validated direct PCR amplification protocol from the reference saliva samples by omitting DNA extraction and quantification steps, which resulted in 80% reduction of the turnaround time. The developed protocol is cost effective, time efficient and it does not compromise with the quality of DNA profiles. To the best of our knowledge, this is the first report for direct amplification of DNA with the most commonly used non-direct multiplex STR kits without any pre-treatment of the sample. Complete DNA profiles matching all the essential quality parameters were obtained successfully from all the tested samples.

Project description:Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.

Project description:Over the past forty years, stable isotope analysis of bone (and tooth) collagen and hydroxyapatite has become a mainstay of archaeological and paleoanthropological reconstructions of paleodiet and paleoenvironment. Despite this method's frequent use across anthropological subdisciplines (and beyond), the present work represents the first attempt at gauging the effects of inter-laboratory variability engendered by differences in a) sample preparation, and b) analysis (instrumentation, working standards, and data calibration). Replicate analyses of a 14C-dated ancient human bone by twenty-one archaeological and paleoecological stable isotope laboratories revealed significant inter-laboratory isotopic variation for both collagen and carbonate. For bone collagen, we found a sizeable range of 1.8‰ for δ13Ccol and 1.9‰ for δ15Ncol among laboratories, but an interpretatively insignificant average pairwise difference of 0.2‰ and 0.4‰ for δ13Ccol and δ15Ncol respectively. For bone hydroxyapatite the observed range increased to a troublingly large 3.5‰ for δ13Cap and 6.7‰ for δ18Oap, with average pairwise differences of 0.6‰ for δ13Cap and a disquieting 2.0‰ for δ18Oap. In order to assess the effects of preparation versus analysis on isotopic variability among laboratories, a subset of the samples prepared by the participating laboratories were analyzed a second time on the same instrument. Based on this duplicate analysis, it was determined that roughly half of the isotopic variability among laboratories could be attributed to differences in sample preparation, with the other half resulting from differences in analysis (instrumentation, working standards, and data calibration). These findings have serious implications for choices made in the preparation and extraction of target biomolecules, the comparison of results obtained from different laboratories, and the interpretation of small differences in bone collagen and hydroxyapatite isotope values. To address the issues arising from inter-laboratory comparisons, we devise a novel measure we term the Minimum Meaningful Difference (MMD), and demonstrate its application.

Dataset Information

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

Aim

Methods

Results

Conclusion

Publications

STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets