Fundamentals of Forensic DNA Typing , Fundamentals of Forensic DNA Typing , کتابخانه دیجیتال جندی شاپور اهواز

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Fundamentals of Forensic DNA Typing

DNA analysis is frequently used to acquire information from biological material to aid enquiries associated with criminal offences, disaster victim identification and missing persons investigations. As the relevance and value of DNA profiling to forensic investigations has increased, so too has the desire to generate this information from smaller amounts of DNA. Trace DNA samples may be defined as any sample which falls below recommended thresholds at any stage of the analysis, from sample detection through to profile interpretation, and can not be defined by a precise picogram amount. Here we review aspects associated with the collection, DNA extraction, amplification, profiling and interpretation of trace DNA samples. Contamination and transfer issues are also briefly discussed within the context of trace DNA analysis. Whilst several methodological changes have facilitated profiling from trace samples in recent years it is also clear that many opportunities exist for further improvements.

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Forensic trace DNA: a review

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA.

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters.

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Mystery solved: the identification of the two missing Romanov children using DNA analysis.

This article reviews what is currently known about short tandem repeat (STR) allelic sequence variation in and around the twenty-four loci most commonly used throughout the world to perform forensic DNA investigations. These STR loci include D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, SE33, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045. All known reported variant alleles are compiled along with genomic information available from GenBank, dbSNP, and the 1000 Genomes Project. Supplementary files are included which provide annotated reference sequences for each STR locus, characterize genomic variation around the STR repeat region, and compare alleles present in currently available STR kit allelic ladders. Looking to the future, STR allele nomenclature options are discussed as they relate to next generation sequencing efforts underway.

STR allele sequence variation: Current knowledge and future issues.

A multiplex PCR was designed for the loci D2S1338, D16S539, D18S51, TH01 and FGA using redesigned primers in order to reduce the lengths of the amplification products compared to the designs used in commercially available multiplex PCR kits, also including amelogenin. The new PCR primers were used to amplify highly degraded DNA from casework samples, which had shown no or only poor results for these loci in previous analyses with standard primer sets. The application of the new miniSTR-multiplex resulted in an increased overall typing success rate for degraded DNA samples. In a concordance study between the conventional and the newly designed primers, no genotype differences were revealed in 124 randomly selected individuals.

A new “miniSTR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA

Unusually large variant alleles were observed in the short tandem repeat (STR) systems D3S1358 and D21S11, both of which are included in the international standard set of loci (ISSOL) and routinely typed in National DNA intelligence databases worldwide. The observed alleles fell within the size range of the adjacent STR marker, which could easily cause problems with respect to correct allele assignments for both loci concerned. We compared the amplification and potential interpretation with three different commercially available kits, which are frequently used in forensic work. PCR products were cloned and sequenced in order to determine the structure of these unusual allele variants and confirm their size and designation (D3S1358 allele 26, D21S11 allele 46). In the locus D21S11 we observed an as yet undescribed partial duplication of the constant region.

R. Mühlmann

Papers

A new “miniSTR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA

Unusual variant alleles in commonly used short tandem repeat loci.