Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken

Gene technology using polymerase chain reaction (PCR) has markedly advanced in recent year and has been introduced in clinical laboratories. In this paper, the genotypes of genomic DNAs of subjects with cisAB blood group were analysed using three methods, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and the PCR-direct sequencing method, and directly determined using the polymerase chain reaction (PCR) amplification of specific alleles (PASA)-method. The differences among the methods were as follows, PCR-RFLP and PCR-direct sequencing method require 2-step procedures, and are complicated for clinical laboratories. The PASA method is based on the fact that PCR amplification occurs only when the 3' endbase of the primer is matched to sites of the nucleotide substitution of ABO allelic cDNA. Three of five regions of allelic DNAs were co-amplified in a single PCR (multiplex-PCR) in this study. ABO and cisAB blood group genotypes were directly determined, based on the molecular size of allele-specific amplification products. The PASA method requires only about 4 hours from starting PCR to results, making it rapid, simple and useful for detecting the genotype of ABO and cisAB blood groups in comparison with PCR-RFLP and the direct sequencing methods and will allow this procedure to be very versatile and widely used throughout the research and clinical diagnostic communities. The analyses of the nucleotide sequence at nucleotides No. 261, 526, 703, 796 and 803 in 3 major subjects in the cisAB blood group (cisA2B3, cisA1B3 and cisA2B) revealed chimeric structures of the A allele and B allele on the same gene.