Determination of cag repeat length in the androgen-receptor gene using frozen serum

Abstract

Objectives To determine CAG repeat length in exon one of the androgen-receptor gene, using frozen serum as a source of deoxyribonucleic acid (DNA). Methods Samples from previously frozen serum samples were prepared for polymerase chain reaction (PCR) by heating thawed serum to 100 °C and subsequent centrifugation at 16,000 g. Three microliters of supernatant were added to the PCR in combination with primers flanking the CAG repeat in exon one of the human androgen-receptor gene. After 35 cycles, the PCR amplicons were electrophoresed on an agarose gel, excised, purified, and sequenced. CAG repeat length was directly determined from DNA sequencing gels. Results Amplified DNA fragments were obtained after PCR from 140 of the 200 specimens examined to date. The DNA amplicon was successfully sequenced in 111 samples derived from 104 individuals. Within this group, the androgen receptor CAG repeat length varied from 11 to 27. The median CAG repeat length was 21 ; the lower and upper quartiles were 18 or less and 23 or more, respectively. Results were consistent in each case that CAG repeat length was repetitively determined. Conclusions The establishment of a methodology to determine androgen-receptor gene CAG repeat length from frozen serum samples opens multiple new opportunities to explore the potential clinical significance of this genetic polymorphism.