Distribution of mtDNA Haplogroups in a Population Sample from Poland

Abstract

DNA was isolated using standard organic method followed by double phenol/chloroform/isoamyl alcohol extraction and its concentration was determined fluorescently with PicoGreen on Fluoroscan Ascent Fl. Amplification was performed in two 8-plex reactions. PCR mixture consisted of 5μL of Qiagen Multiplex PCR Master Mix, 1μL of premixed primers (1), 1–4μL of DNA extract (about 0.5 ng of DNA) and distilled water up to 10μL. Reaction conditions were as follows: pre-incubation step: 95◦C for 15 min; denaturation: 94◦C for 30 s, annealing: 60◦C for 90 s, extension: 72◦C for 90 s; 30 cycles; final extension: 72◦C for 10 min. The samples were amplified using GeneAmp PCR System 9700 or 9600 (PE Applied Biosystems). PCR products were detected by 3% agarose gel electrophoresis. PCR primers and unincorporated dNTPswere removed byMicroconeYM-100Centrifugal Filter Devices (Millipore) according to manufacturer’s protocol. SNPs were detected in two multiplex minisequencing reactions. Each reaction