Abstract
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.
Highlights
Polymorphic CAG repeats in the human androgen receptor gene (HUMARA), located at Xq12, have been used as a highly informative genetic marker for human female tumor cells
We show that a fluorescent loop-hybrid mobility shift (LH-MS) technique facilitates the detection of far more complex genotypic variants of CAG repeats ranging from 17 to 31 in repeat numbers
CAG repeat length polymorphism in HUMARA exon 1 was first analyzed in male DNA
Summary
Polymorphic CAG repeats in the human androgen receptor gene (HUMARA), located at Xq12, have been used as a highly informative genetic marker for human female tumor cells. Because of the random inactivation of one X chromosome, the heavily methylated state of one of the heterozygous alleles provides a useful tool, together with methylation-sensitive restriction enzymes, to examine clonality of tumor cells in female cancer patients [1,2,3]. In this respect, a simple method to determine the allelic status of CAG repeats using slab-gel electrophoresis would be of some value. The present technique is capable of detecting even a single repeat unit difference and reveals heterozygosity of this locus in 87% of human female study population
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