Detection of nucleotide variations in the D-loop region of bovine mitochondrial DNA using polymerase chain reaction-based methodologies.

Allele-specific polymerase chain reaction (ASPCR) and single-strand conformation polymorphism (SSCP) were used to characterize 16 Holstein maternal lines. ASPCR was used to detect polymorphic nucleotides at eight different positions in the displacement loop (D-loop) of bovine mitochondrial DNA (mtDNA): 16022, 16057, 16074, 16231, 16247, 106, 169 and 363. ASPCR analysis of the maternal lines showed variations at nucleotide positions 106, 169, and 363 of the mtDNA. Within-line variation was observed in five maternal lines for nucleotide 363. SSCP analysis of the mtDNA D-loop region revealed variations that classified the maternal lines into six different genotypes. Based upon the variations observed by ASPCR and SSCP analysis, the animals representing the 16 maternal lines could be assigned to 10 different genotypic groups. These procedures provide a rapid, simple, non-radioactive, and reliable method of detecting polymorphism in the D-loop region of bovine mtDNA.