PCR was performed on “DNA Engine Dyad” (Bio-Rad, The Netherlands) with 1.25 U of “SmarNGTaq” DNA-polymerase (Dialat Ltd., Moscow, Russia); 1× “Ampli” PCR-buffer (Dialat Ltd., Moscow, Russia); 1.2 M Betaine (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); 2.5 mM MgCl2 solution (Dialat Ltd., Moscow, Russia); and dNTPs (0.2 mMdATP, 0.2 mMdTTP, 0.2 mMdCTP, and 0.2 mMdGTP; Dialat Ltd., Moscow, Russia). The primers were as follows: 0.4 mM Rep1F 5′- TCCCACCCTCAGGCTCCTC-3′; 0.4 mM Rep1R1seq 5′-AGTGCAGACCAGGGCGCCG-3′. The PCR conditions consisted of an initial “hot-start” at 95 °C for 3 min, followed by 40 cycles: 95 °C—30 s, 62 °C—5 s, and 72 °C—15 s. The PCR product was 351 bp for chromosome 4q35 and 345 bp for chromosome 10q26.
Sanger sequencing of PCR-products was performed by the “DNA-diagnostic laboratory” (Moscow, Russia) on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The obtained sequences were analyzed with FinchTV v.1.5 software.
Sanger sequencing of PCR-products was performed by the “DNA-diagnostic laboratory” (Moscow, Russia) on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The obtained sequences were analyzed with FinchTV v.1.5 software.