NCJ Number
186700
Journal
Legal Medicine Volume: 2 Issue: 1 Dated: March 2000 Pages: 15-20
Date Published
March 2000
Length
6 pages
Annotation
The inverse PCR technique was applied to type three major alleles (A1, B, and O1) of the ABO blood group by simultaneously detecting separated allele-determining nucleotides (the 261st based in exon 6 and the 796th and 803rd nucleotides in exon 7) of the ABO gene.
Abstract
The study amplified a sequence of approximately 1.7 kb from exons six to seven of each allele, both termini of the fragment ligated, and allele-typing performed by the inverse PCR-restriction fragment length polymorphism (IP-RFLP) and allele-specific inverse-PCR (ASIP) methods. For intramolecular ligation, primers for the first PCR were designed to have Acc I-restriction sites within the sequences, and both termini of the 1.7-kb fragment were digested with Acc I. Using the IP-RFLP method, the inverse PCR product was digested with Kpn I, Nla III, and Dde I. The A1, B, O1-standard (Oa) and O1-variant (Og) alleles were detected as 365-bp, 124-bp, and 128-bp fragments, respectively. By the ASIP method using four allele-specific primers, 222-bp, 124-bp, and 232-bp fragments were amplified from A1, B, and O1 templates, respectively. Inverse PCR, which involves the ligation of separated regions, was explored to analyze unknown sequences that flanked a region of known sequence. The authors applied this technique to analyze separated allele-determining sequences in the genes of the MN11 and ABO blood group systems. This technique may also be applicable to the genotyping of other blood groups and haplotyping of linked polymorphisms. 3 figures and 20 references