New class I allele of the insulin-linked polymorphic region (ILPR) detected by PCR amplification

Abstract

The insulin-linked polymorphic region (ILPR) located on the 5′ flanking region of the human insulin gene (INS), is considered to be an important transcriptional regulatory region or the susceptibility region of insulin-dependent diabetes mellitus (IDDM). The polymorphism is generated by variation in the number of repeat units within a given ILPR, which determines its overall length, and by minor nucleotide sequence heterogeneity within the individual repeats. Because of high G+C content or highly repetitive sequences in the ILPR, it makes them less amenable to PCR analysis. In this study, we amplified in vitro the ILPR using the PCR mixture containing 40% and 60% 7-deaza-dGTP, a nucleotide analog of dGTP, in stead of the standard PCR mix. The amplified DNA contained an ILPR of 572 bp, indicating a typical class I allele that contains 40 directly repeated oligonucleotides and revealed high G+C content of 72. 4%. The arrangement of the repeat units of the ILPR was different from the six ILPR alleles reported so far. Thus, this DNA sequence can be considered as 7th ILPR allele or 5th class I allele. Therefore, a modified PCR procedure using 7-deaza-dGTP permits to amplify easily or to characterize the class I allele linked to IDDM susceptibility.