Fortgeschrittene Techniken in Biologie und Medizin
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Analysis of Human Androgen Receptor Polymorphism Using Fluorescent Loop-Hybrid Mobility Shift Technique

Tomokazu Ishikawa, Shoichi Matsukuma, Mitsuyo Yoshihara, Tatsuo Kurosawa and Yohei Miyagi

A fluorescent loop-hybrid mobility shift (LH-MS) technique was introduced for the analysis of polymorphic sites in exon 1 of the human androgen receptor gene. (CAG)17-31 or (CTG)17-31 loops were assumed to protrude from the sense- or antisense strands of the PCR products following hybridization with reverse or forward LH probes, respectively. When an array of male DNA was analyzed using Cy5-labeled LH probes, a unique linear correlation was established between CAG repeat lengths and the fluorescent LH band positions on polyacrylamide gels. This linearly shifting band patterns were used to assemble LH ladder size markers of CAG repeat lengths. Analysis of female DNA revealed that 87% of females are heterozygous for CAG repeat polymorphisms, which could be informative for clonality analysis of tumors in female cancer patients. As a proof of principle, heterozygous female colorectal tumor DNA was examined with fluorescent LH-MS technique and loss of one allele after treatment with methylation-sensitive restriction enzyme HpaII was clearly exhibited.