Low Template DNA: Tad, Touch and Traces

Small amounts of DNA, typically less than 100 pg, termed Low Template (LT) DNA which is extremely useful in forensic casework. The current advancement of highly sensitive multiplex PCR systems consisting of short tandem repeat (STR) markers have allowed the development of genetic profiles from much lower quantities of DNA, from such samples. However, this sensitivity of the STR profiling systems also leads to issues of secondary transfer of LT DNA such as the implication of innocent parties as a result of background contamination and profiles having artefacts due to stochastic effects. This review explores the varying deposition mechanisms, technological advancements in both collection and analysis, and ultimately evaluates the usefulness of LT DNA considering its admissibility as forensic evidence

A Molecular Study of Pakistani Populations Using Short Tandem Repeat Markers

In order to implant DNA typing methods into the Pakistani medico legal system it was necessary to study the genetic structure of the Pakistani populations. For this purpose seven hundred and sixty fresh blood and buccal swab samples were collected from seven ethnic groups the Punjabi, Pushtoon, Sindhi, Baluchi, Makrani, Brosho & Kalash. DNA extraction from the samples was performed using the PuregeneRTM kit and ChelexRTM extraction methods. The populations were profiled for three autosomal STR loci D3S1358, vWA and FGA for which modified primers were designed and allelic ladders prepared. Some of the samples were profiled using the AmpFlSTRRTM Blue kit. A database of the allele/genotype frequencies was established and the structure of the population was studied. It was established that the allele and genotype frequencies differ significantly among the Pakistani populations. In the Punjabi and Sindhi populations the exact test p value was 0.05. Substructure was detected in these populations and the FST values of 0.01-0.04 were determined between different populations in pairwise comparisons. Using a simulation study it was shown that incorporation of the substructure in the calculation of allele and genotype frequencies results in conservative estimates of the likelihood ratio of match. The analyses favoured the generation of separate databases for sub populations even if they are as closely related as are the Pakistani populations. The second part of the project was to study the populations using the Y chromosome specific STR systems. For this purpose seven Y STRs (DYS19, 3891, 389II, 390, 391, 392 & 393) were selected which define the haplotype Yh1 (Y chromosome haplotype I). Sequenced allelic ladders were prepared for all the loci. The different ethnic groups of Pakistan were profiled for these loci in two multiplex reactions and 564 complete haplotypes generated. The individual locus and haplotype frequencies were calculated for all the populations. Yh1 had a high diversity and discrimination capacity for the Pakistani populations when these populations were compared to other populations. The diversity within and between populations was determined by AMOVA. It was established that diversity for Y STRs within the populations was greater than between populations. AMOVA also revealed that there was much less diversity between the three major populations of Pakistan pointing to closer male lineage relationship between these populations. Two smaller ethnic groups the Brosho and the Kalash generated higher diversity when compared to the major populations of Pakistan. Phylogenetic trees (UPGMA & NJ) were constructed using the phylogenetic software PHYLIP. The phylogenetic analysis of the Pakistani populations suggested that the Punjabi and the Sindhi populations are closest to each other as are the Baluchi and the Makrani populations while the positions of the Kalash and Brosho populations were mostly distant from those of the other Pakistani populations

Are Roma People Descended from the Punjab Region of Pakistan: A Y-Chromosomal Perspective

Gypsies are a separate ethnic group living in Pakistan and some other countries as well. They are mostly known as ‘Roma’ and ‘untouchables’. They have different types of lifestyles as compared to other common people, as they always keep migrating from one place to another. They do not have proper houses; they live in tent houses and most probably work on daily wages to earn their living. Gypsies cannot be specified according to the place of residence and can only be classified according to their migration route. Previous historical and linguistic research showed the north Indian origin of Roma people. The present study collected 285 unrelated Roma individuals living in Punjab and typed with the Goldeneye Y20 system. Allelic frequencies ranged between 0.0035 and 0.5266, with haplotype diversity (HD) of 0.9999 and discrimination capacity (DC) of 0.8790. Gene diversity (GD) ranged from 0.6489 (DYS391) to 0.9764 (DYS391) (DY385ab). A total of 223 unique alleles were observed. Interestingly, the haplogroup R accounted for 40.56% and J for 22.06%. In MDS analysis, Pakistani Roma formed a close cluster with Roma from Constanta, Romania. The migration pattern of the Roma population from Pakistan, India and Europe was inferred using coalescence theory in the Migrate-n program. Overlapping Y-STR data were used to test different migration models. These migration models showed us the dominant gene flow from Pakistan to India and Europe to Pakistan. The results of our study showed that Y STRs provided substantially stronger discriminatory power in the Pakistani Roma population

Online calculator for individual affiliation to a major population group

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15–17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator (http://cracs.fc.up.pt/popaffiliator) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification

Increasing the reference populations for the 55 AISNP panel: the need and benefits

Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples (with >1100 individuals studied) to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful

Analysis of rpoS and bolA gene expression under various stress induced environments in planktonicand biofilm phase using 2-ΔΔCT method

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5–6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H2O2) as compared with rpoS

Evaluation of five DNA extraction systems for recovery of DNA from bone

Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch® gDNA Plant Kit (Life Technologies), DNA IQ® System Kit (Promega), DNeasy® Blood & Tissue Kit (Qiagen) and PrepFiler® BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq® qPCR Master Mix (Promega) using an Applied Biosystems® 7500 Real-Time PCR System and the extracts were amplified using an inhouse multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch® gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition

Evaluation of decalcification for recovery of DNA from bone

The effect of decalcification on pulverised bone samples was studied to evaluate its value when processing reasonably good quality samples. The decalcified and non-decalcified bone samples were extracted using phenolchloroform extraction method, quantitated with GoTaq® qPCR Master Mix (Promega®, UK) using Applied Biosystems® 7500 Real-Time PCR System (Thermo Fisher Scientific®, UK) and amplified using an in-house amplification multiplex. The results showed that the decalcification of bone samples is not a necessary when processing good quality samples and higher DNA yields were obtained without the decalcification process. The electropherograms produced from the extracted DNA samples without decalcification were good quality and displayed no PCR inhibition

The AMOVA analysis phylogenetic relationships of Pakistani populations using Y STRs

Pakistan is a large country with a diverse population consisting of four significant population groups besides many smaller isolated populations. Male population samples were collected from indigenous populations and the extracted DNA was used to amplify seven Y STRs. Allele frequencies within each population were similar as were the haplotype frequencies. The data showed that individual populations were diverse in spite of a high rate of consanguineous marriages. This work indicates that, even with smaller number of loci, significant amount of genetic and phylogenetic information can be gained from the data.status: publishe

Involvement of bolA and rpoS genes in oxygen uptake (Respirometry) by E. coli K-12 MG1655 under various stress conditions

Differences in respiratory activity of Escherichia coli under various stress-induced environments has shown the ability of rpoS and bolA genes to respond in several stress conditions and adaptation, leading to an increase rate of oxygen uptake. The results have shown the rate of respiratory activity (defined in mg O2/g bacteria min-1) changed under various stress-induced conditions, i.e. heat shock, cold shock and oxidative stress. Sudden shifts from optimal growth conditions to stress-induced environments led to an increase of the oxygen uptake rate of E. coli in presence of rpoS and bolA genes