Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA

Abstract

A specific segment of mitochondrial DNA from 18 people was examined by two methods of direct DNA sequencing. This segment includes a small noncoding region (V) shown before by restriction analysis to exhibit length polymorphism. All 11 of the human mtDNAs previously reported to have a deletion in this region proved to lack one of the two adjacent copies of a 9-base-pair sequence normally present in human mtDNAs. Phylogenetic analysis suggests that this deletion occurred only once during the evolution of modern types of human mtDNA and that it will be a valuable anthropological marker for peoples of East Asian origin. The one human mtDNA reported to have an addition in region V differs from the wild type by two mutations in the first copy of the 9-base-pair sequence: one transition and an addition of four cytosines, thereby producing a run of 11 cytosines. One of the direct DNA sequencing methods uses a single oligonucleotide primer to facilitate dideoxy sequencing from purified mtDNA templates. The second, more successful, method first amplifies this mtDNA segment enzymatically with two flanking primers (the "polymerase chain reaction") and then uses a third primer for DNA sequencing. This latter method, which works on the DNA extracted from small amounts of blood as well as on purified mtDNA, is shown to be a rapid means of defining sequence variants without purifying and cloning the same DNA segment from many individuals.

Identification of differentially expressed genes in skeletal muscle of non-diabetic insulin-resistant and insulin-sensitive Pima Indians by differential display PCR

“…The polymorphic MTCO2 cDNA bands resulted from insertion/ eletion of a 9-base pair repeat sequence in position 8271-8290 of the mitochondrial genome, which is common in Asians, Polynesian and Native Americans [37,38,39]. None of the genotyped polymorphisms were related to insulin sensitivity in the 50 Pima subjects.…”

Mitochondrial DNA polymorphisms in Yunnan nationalities in China

“…For each population, the frequency of the deletion was 25% for the Lahu, 19% for the Dai, and 5% for the Wa; however, in Tibetans, the 9-bp deletion was completely absent, indicating the unique genetic feature of this ethnic group. Some studies have suggested multiple origins for the deletion in Asia (Schurr et al 1990;Ballinger et al 1992;Redd et al 1995), although the deletion may have occurred only once during the evolution of modern types of human mtDNA (Wrischnik et al 1987). Also, a comparison of the D-loop region sequence data from Asian and sub-Saharan African populations with the deletion indicated that the 9-bp deletion arose independently in subSaharan Africa and Asia, and that the deletion has arisen more than once in Africa (Soodyall et al 1996).…”

Maori origins, Y-chromosome haplotypes and implications for human history in the Pacific

“…The presence of the 9-bp deletion in the mtDNA region V was determined by PCR, followed by gel electrophoresis with ethidium bromide visualization, as described in Wrischnik et al [1987]. The European and Asian mtDNA haplogroups were evaluated as described in Macaulay et al [1999].…”