Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination

Abstract

The high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA material for further analysis. Furthermore, human hypervariable regions - HV1 and HV2, located on mtDNA were sequenced to define human haplogroups. For extraction, samples of buccal mucosa from ten human volunteers were taken and three different protocols were used (a method with ammonium acetate, a salting-out method and GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific)). Concentration and purity of DNA were measured on BioPhotometer. Sequencing was performed by Sanger method on 3130 Genetic Analyzer (Applied Biosystems) with commercial BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were processed using Chromas and MEGA software and compared with sequence databases with BLAST tool on NCBI. Haplogroups and mutations were determined in the MITOMAP online database. Results of extraction indicated that the Purification Kit gives the highest concentration of DNA (11.86±0.63) and low purity (1.17±0.02), while a method with ammonium acetate gave the best purity (1.67±0.17). Sequencing obtained on DNA extracted with a commercial kit, gave 5 different human haplogroups U2e, T1a, J1c, H2a, K. In conclusion, it is recommended to use the Purification Kit for obtaining high integrity DNA that can be used for effective sequencing. Determination of haplogroups and haplotypes can reveal our ancestry and have important implications in molecular systematics, phylogenetics and phylogeography.