Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

Amplification of human mitochondrial DNA (mtDNA) has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII) were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies

Genomic DNA from rat blood: A comparison of two extraction methods

In this study, two methods for DNA extraction from fresh rat blood were compared. One is based on the use of cetyltrimethylammonium bromide (CTAB method), while the other one is well-known salting out method. Spectrophotometric analysis was employed to assess yield and purity of isolated DNA, while agarose gel electrophoresis was carried out to evaluate DNA integrity. The results have clearly demonstrated that the extraction method has significantly influenced the quantity and purity of isolated DNA. By using the CTAB method, a larger quantity of high-molecular weight DNA with good purity is obtained which, along with time- and cost-efficiency of the procedure, makes this method more suitable for the extraction of DNA from rat whole blood

Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

Amplification of human mitochondrial DNA (mtDNA) has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII) were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies.[Acknowledgments. This work was supported by the Ministry of Education and Science of the Republic of Serbia (Grant no. III 47025).

Allele frequencies and forensic parameters of 22 autosomal STR loci in a population of 983 individuals from Serbia and comparison with 24 other populations

Background Analysis of allele frequencies of short tandem repeat (STR) loci in ethnically diverse populations is essential for forensic reference database construction and studies on population genetics. Aim To analyse genetic polymorphisms of 22 autosomal STR loci in the Serbian population and to compare them with previously published data from some European and Turkish populations. Subjects and methods The study was conducted among 983 unrelated individuals from Serbia. Genotyping was performed using the PowerPlex® Fusion amplification kit. Allele frequencies and forensic parameters were calculated using FORSTAT software. Interpopulation comparisons and genetic distance calculations were performed in Arlequin and POPTREEW software. Results A total of 280 alleles were detected with corresponding allelic frequencies ranging from 0.0005 to 0.5255. Based on heterozygosity and the polymorphism information content, D1S1656 and Penta E may be considered as the most informative markers. Both the combined power of discrimination (CPD) and the combined power of exclusion (CPE) for the 22 analysed loci were higher than 0.999999. The combined match probability (CPM) for all 22 loci was 6.773688e−29. Conclusion With respect to the results, the 22 STR loci are highly polymorphic and discriminating in the Serbian population and could be used for forensic practice and population genetics studies

Synthesis, Swelling Properties and Evaluation of Genotoxicity of Hydrogels Based on (Meth)acrylates and Itaconic Acid

In this study we prepared hydrogels based on 2-hydroxyethyl methacrylate (HEMA): PHEMA homopolymer and two terpolymers of HEMA, itaconic acid (IA) and two poly(alkylene glycol) (meth) acrylates (PAGM): poly(ethylene glycol)(6) acrylate (P(HEMA/IA/PAGM1)) and poly(propylene glycol)(5) methacrylate (P(HEMA/IA/PAGM2)). Hydrogels were synthesized by gamma-irradiated radical polymerization and subjected to swelling measurements and genotoxicity evaluation. Swelling studies confirmed that these hydrogels deserve consideration as biomaterials due to their ability to swell in phosphate buffer but maintaining physical integrity for a prolonged contact time after equilibrium state has been reached. Comet assay showed certain genotoxic effect following cell exposure to extracts of hydrogels, which was dependent on the concentration of extracts, chemical composition of hydrogels and the degree of crosslinking. The influence of concentration on genotoxicity was the most pronounced. The synthesis of these novel HEMA-based hydrogels should be optimized so as to reduce their toxicity and enable the use in clinical practice