Genotyping TERT VNTR-MNS16A Polymorphism

The presence of the VNTR-MNS16A TERT gene polymorphism was assessed in BC patients and in healthy women by PCR amplification followed by electrophoresis in sequencing gel, as described by Wysoczanska et al. [41 (link)]. PCR was performed in a 2720 Thermal Cycler instrument (Applied Biosystems, Foster City, CA, USA) using the forward and reverse primer sequences (5′-AGGATTCTGATCTCTGAAGGGTG-3′ and 5′-TAMRA-TCTGCCTGAGGAAGGACGTATG-3′) prepared by Genomed (Warsaw, Poland). The amplification procedure included an initial denaturation step for 5 min at 95 °C, followed by 35 cycles: 30 s at 95 °C, 30 s at 65 °C, 30 s at 72 °C and a final extension step for 10 min at 72 °C. The PCR products were diluted with formamide and a GeneScan™500 ROX™ dye Size Standard (Applied Biosystems, Foster City, CA, USA). The samples were denatured at 95 °C for 5 min and analyzed on the 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with an eight-capillary system filled with POP7 polymer (Applied Biosystems, Foster City, CA, USA). The alleles were identified using the GeneMapper software version 4.2 (Applied Biosystems, Foster City, CA, USA).