Genescan 500 tamra

The method for analyzing the CAG repeat polymorphism in the AR gene has been described in detail previously.11 (link)12 (link) Briefly, exon 1 of the AR gene was amplified using the forward 5’-TCCAGAATCTGTTCCAGAGCGTGC-3’ and reverse 5’-GCTGTG-AAGGTTGCTGTTCCTCAT-3’ primers flanking the CAG repeat. One of the primers was marked with Cy5.5 fluorescent dye. Amplification was performed in a 25 μl reaction volume containing 50 ng of genomic DNA and 200 μmol l⁻¹ of each deoxynucleotide triphosphate. The concentration of the primer was 1.2 μmol l⁻¹. PCR conditions were set as follows: 30 cycles of 95°C for 45 s, 56°C for 30 s and 72°C for 30 s for amplification. The PCR program was initiated with a denaturation step at 95 °C for 5 min and terminated with an extension step at 72°C for 5 min.13 (link) The PCR products were analyzed on a Genescan-run ABI 377 DNA gel-slab electrophoresis sequencer (Perkin-Elmer, Co, Norwalk, CT, USA) with a TAMRA-labeled internal length standard (Genescan-500 TAMRA, Applied Biosystems, Foster City, CA, USA). Genotyper software was used to determine the genotypes (Genotyper version 2.0, Applied Biosystems).14 (link)