Sequencing the mitochondrial ATP6 synthase gene: comparing variations in Limulus and Argopecten to human SNPs

Abstract

Mt‐ATP6 is a highly conserved gene belonging to the ATPase family, a group of enzymes required for cellular respiration in the electron transport chain. Human mutations in this gene result in neuropathy, ataxia, retinitis pigmentosa, and Leigh Syndrome. Target organisms: Argopecten irradians and Limulus polyphemus, are declining in Long Island Sound (LIS) respectively affecting the fishing industry and the availability of Limulus blue blood used in medicine.After extracting DNA from Limulus hemocyanin and Argopecten adductor muscle, two sets of overlapping primers were designed to encompass the approximate 700 base pairs of the ATP6 gene using the mitochondrial genome GenBank entries (NC_003057 and NC_012977) as templates. PCR protocols were constructed accordingly; subsequent electrophoresis revealed appropriate products which were then purified, diluted and quantitated using a biophotometer.At this writing, the primer sets will be further diluted to sequencing concentrations and the 38 templates of more than 400 base pairs each will be sequenced using the school's ABI Prism 310 Genetic Analyzer, a single capillary automated sequencer. Single nucleotide polymorphisms (SNPs) and translated protein sequences will be compared to the control GB entries, to each other, and to the human mt‐ATP6 gene using NCBI, ClustalW, the SNP Database, and MITOMAP data files.Women's Seamen's Friend Society of CTApplied BiosystemsWhatmanNOAA, Milford CTCarmela Cuomo, Ph.D