Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs,assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.     

Failed PCR amplifications of MBP-STR alleles due to polymorphism in the primer annealing region

The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed pair of primers we present evidence that this is due to failed amplifications caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described here no exclusions were found (out of 59 mother/child pairs analysed) while one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) from North Portugal are compared. In this sample 13 individuals typed as homozygotes with the pair of primers originally described, were found to be heterozygous when the amplifications were performed with the new reverse primer. By sequence analysis, a substitution in the reverse primer binding sequence originally described was determined. This substitution is located upstream from the repetition site and consists of G→A transition. This variation reaches polymorphic frequency and is responsible for the relatively frequent null alleles due to failed amplifications when the previously designed primers are used.     

6个Y染色体STR基因座遗传多态性及其在亲子鉴定中的应用

目的为研究中国南方汉族人群DYS393等6个Y-STR基因座的遗传多态性并用于法医学鉴定。方法采用PCR复合扩增和基因测序仪荧光检测方法,检查204个无关男性个体,调查南方汉族的6个Y-STR基因座的单倍型频率,并对93对真父子和38对非父子的亲子鉴定样本进行检测。结果DYS393基因座检出5个等位基因,DYS19基因座检出6个等位基因,DYS385Ⅱ基因座检出8个等位基因,DYS390基因座检出6个等位基因,有1DYS391基因座检出4个等位基因,DYS385基因座检出44个等位基因,共检出177种单倍型。93对真父子中,观察到2例分别有1个基因座突变。检测38对非父子,有1个或2个Y-STR基因座排除的案例各有1例(2.6%);有3个和3个以上的Y-STR基因座可以排除父子关系的案例为35例(92.1%);6个Y-STR基因座不能排除父子关系的为1例。结论本研究中的6个Y-STR基历座具有丰富的遗传多态性,可用于法医学个体识别和亲子鉴定。     

Kinship analysis of skeletal remains from the Middle Ages

Medieval cemeteries Klisa-Guca Gora, Alihodze and Glavica-Han Bila located in the Travnik area (Travnik, Bosnia and Herzegovina) were archaeologically examined in the period 2011–2014, revealing human skeletal remains of 11 individuals in total. Archaeological skeletal samples, previously deposited in Travnik Homeland Museum (Travnik, Bosnia and Herzegovina) were subjected to genetic analysis. The aim of this research was to test familiar relationship of 11 individuals excavated from three medieval cemeteries and to predict Y-haplogroup for male individuals. In order to perform molecular-genetic characterisation of collected human skeletal remains, two systems of genetic markers were analysed: autosomal and Y-STR loci. Complete or partial data obtained by autosomal STR typing of 11 individuals were subjected to kinship analysis. Male sex was determined in eight samples out of 11. Direct relatives of the "brother-brother" type were detected in one case with high kinship probability (KP) value of 99.99996 %. Complete or nearly complete and usable Y-STR profiles were obtained for six out of eight male individuals. The presence of identical haplotypes at Y-STR loci and results of Y-haplogroup prediction suggest that all male individuals share the same paternal lineage and belong to J2a haplogroup. Overall, this study emphasises the usefulness, efficiency and sensitivity of STR markers in the molecular-genetic characterisation of old skeletal remains as well as the importance of employing additional markers like Y-STRs in archaeogenetic studies, besides traditionally used autosomal STR markers, in order to get a comprehensive information about close and distant relatives, and ancestry.