High-frequency DNA sequence polymorphisms in the insulin receptor gene detected by denaturing gradient gel blots.

A limiting factor in the study of genetic determinants of human disorders is the availability of informative DNA markers. In this report, we describe an application of the denaturing gradient gel blot method for detecting high-frequency DNA sequence polymorphisms in the human insulin receptor locus. Using two restriction enzymes and cDNA probes for the insulin receptor, we found five DNA polymorphisms. The probe that contained exons 4-10 of the insulin receptor gene detected two two-allelic polymorphisms in HinfI digests, one at denaturant concentrations of 38%/39% and the other at 46%/48%. The probe that contained exons 14-22 detected three two-allelic polymorphisms in Sau96I digests, the first at denaturant concentrations of 34%/35%, the second at 38%/39%, and the third at 46%/47%. All these DNA polymorphisms segregated in families in a Mendelian fashion, and the allelic distribution for each of them did not deviate from Hardy-Weinberg equilibrium. The identified polymorphisms were in linkage equilibrium and provided sufficient genetic information to determine parental haplotypes at the insulin receptor locus in small two-generation families. The denaturing gradient gel blot method is a very sensitive technique for identifying sequence polymorphisms in genomic DNA; its application will facilitate the search for genes involved in the development of many inherited disorders.