Journal of Clinical Endocrinology & Metabolism, Vol 74, 1027-1031, Copyright © 1992 by Endocrine Society

ARTICLES

Detection of mutations in the human insulin gene by single strand conformation polymorphisms

M Kishimoto, H Sakura, K Hayashi, Y Akanuma, Y Yazaki, M Kasuga and T Kadowaki
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.

Single strand conformation polymorphisms (SSCP) is the method by which mutations can be detected in DNA amplified by the polymerase chain reaction (PCR). This method utilizes the fact that the electrophoretic mobility of single stranded DNA under nondenaturing condition depends not only on its size but also on its conformation. We examined whether this technique could detect five mutations that had previously been identified in the insulin genes of patients with abnormal insulin or familial hyperproinsulinemia. Five mutant insulin genes were constructed by site directed mutagenesis, and the cloned mutant insulin genes were amplified by PCR. All five mutations were detected because they were associated with shifts in the electrophoretic mobility of the DNA fragments. In addition, we analyzed amplified genomic DNA from a patient who is heterozygotes for the Insulin Wakayama mutation A3(Val--- -Leu). This mutation was also detected by the SSCP technique. We conclude that PCR-SSCP method is a simple, fast, and efficient method for detection of mutations in the human insulin gene.

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