Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor's alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

STR data for PowerPlex 16 System from Buenos Aires population,Argentina

Allele frequencies for the 15 autosomic STR loci included in the PowerPlex 16 System kit (Promega Corp.) were estimated from a sample of 143 unrelated individuals living in Capital Federal and in Buenos Aires Metropolitan Area, Argentina. Population showed to be in HWE.     

STR data for PowerPlex 16 System from Neuquen population,SW Argentina

Allele frequencies for the 15 autosomic STR loci included in the PowerPlex 16 System kit (Promega) were estimated from a sample of 111 unrelated individuals living in Neuquen province, southwest of Argentina. Population showed to be in HWE.     

Validation of the AMPFlSTR SGM plus system for use in forensic casework

The AMPFlSTR((R)) SGM Plus system is a commercially available STR multiplex produced by Applied Biosystems, a division of Perkin Elmer, Foster City, California, USA that supersedes SGM. The multiplex contains the six SGM loci, amelogenin and four additional loci. These additional loci are D3S1358, D19S433, D16S539 and D2S1338. Consequently, the match probability is significantly improved (conservatively quoted as 1 in 10(9) for reporting a full profile match). The system was subjected to validation. For example, ageing and degradation studies demonstrated semen stains to be the most stable evidence type, whereas buccal scrapes were the least stable. An apparent rise in the sensitivity increases the chance of obtaining successful results from the more difficult samples submitted for analysis. Two of the new loci (D3S1358 and D19S433) are low molecular weight (between 100 and 150 base pairs); this improved the success rates of the degraded samples where high molecular weight loci may drop out. Of 26 non-primates tested, four gave results that appeared as single peaks and were unlikely to cause interpretation problems. None of the 19 micro-organisms tested gave discernible results. Extensive casework and simulated casework studies demonstrated that SGM and SGM plus results were comparable. There was one example of a null allele (primer binding site mutation) recorded at the HUMFIBRA locus (in both systems). However, a concordance study of 1000 samples using both SGM and SGM plus did not demonstrate any differences in typing.     

STR data for the PowerPlex 16 loci for the Chinese population in Hong Kong

Allele frequencies for the 15 STR loci included in the PowerPlex 16 kit were obtained from a sample of 247 unrelated Chinese in Hong Kong.     

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

Validation of the AmpFlSTR Profiler Plus PCR amplification kit for use in forensic casework

According to TWGDAM guideline 4.5 (1), prior to implementing a new DNA analysis procedure or an existing DNA analysis procedure developed by another laboratory, the forensic laboratory must first demonstrate reliability of the procedure in-house. Seven phases were designed to validate the use of the AmpFlSTR Profiler Plus PCR Amplification Kit, as well as the PE Applied Biosystems 310 Genetic Analyzer. This report summarizes the results obtained for each of the seven phases of the validation study which included the following evaluations: polymer, reproducibility, sensitivity, stutter, precision, mixtures and nonprobative casework.     

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.     

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.     

Effect of low-dose radiation on mutation rates of STR loci commonly used in forensic casework

The possible effect of low-dose radiation on STR markers in people exposed to radiation during their professional activities was estimated in this study. We evaluated mutation rates in 17 forensic STR loci typed by the COrDIS-18 kit (CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D21S11, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, and vWA) in 78 families (father–mother–child) with one parent exposed to low-dose radiation before fertilization. Five mutations were observed. In two cases, the new alleles were delivered from the non-exposed parent. The calculated mutation rate for the 17 studied STR loci in families appeared to be in good concordance with data published for normal populations. No evidence for an elevated mutation rate in STR markers after low-dose radiation was found.