Methods for Sequencing the Mitochondrial DNA A+T-Rich Region of Cochliomyia Macellaria(Diptera: Calliphoridae) from North America

Abstract

ABSTRACTThe A+T-rich region (ATRR) is the major non-coding segment of insect mitochondrial DNA (mtDNA). It is a hypervariable locus, and therefore useful for population genetic studies. Forensic entomologists could potentially use the ATRR to test for regional subpopulations of a carrion insect species, or to reconstruct the postmortem movement of a corpse. However, the ATRR presents greater genotyping difficulties compared to the commonly studied mtDNA coding regions. Homomeric stretches and length heteroplasmy within the ATRR, and a lack of conserved internal PCR primers, so that PCR amplicons of around 1500 bp are necessary, make both direct sequencing and sequencing of cloned product difficult. We designed ATRR internal primers, complementary to the same annealing site, and tested them using C. macellarla specimens from Puerto Rico, Florida, Tennessee, and West Virginia. The entire ATRR sequence, other than the primer site, could be obtained from cloned PCR product. The ability to use shorter PCR amplicons should also make this method more suitable for analyzing degraded evidence.