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NCJRS Abstract

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NCJ Number: 222183 Find in a Library
Title: Methods for Sequencing the Mitochondrial DNA A+T- Rich Region of Cochliomyia Macellaria (Diptera: Calliphoridae) From North America
Journal: Canadian Society of Forensic Science Journal  Volume:40  Issue:4  Dated:December 2007  Pages:165-172
Author(s): Petra Boehme; Jeffrey D. Wells
Date Published: December 2007
Page Count: 8
Type: Report (Study/Research)
Format: Article
Language: English; French
Country: Canada
Annotation: This study designed and tested new internal primers that can be used to get additional A+T-rich region (ATRR), the major non-coding segment of insect mitochondrial DNA (mtDNA), sequence data for forensically important North American flies, beginning with C. macellareia, one of the more common blow fly species used as evidence in a death investigation that involves decomposing human remains.
Abstract: Using all four primers and cloned PCR products as a template, it was possible to obtain the entire ATRR sequence other than that of the internal primer annealing site. The annealing site must closely match the primers for them to have worked. Complete ATRR haplotypes were deposited in GenBank (Puerto Rico=EU106650, EU106651; Florida=EU106652; Tennessee=EU106653, EU106654, EU106655, EU106656; West Virginia=EU106657). The direct sequencing reactions failed at repeat regions. Elements, such as long thymine nucleotide stretches (25) cause polymerase slippage and length heteroplasmy (20). Downstream from a length heteroplasmy site, a sequence electropherogram becomes unreadable. Although researchers observed intraspecific variation in the form of slightly different clones from the same individual, these preliminary data suggest it is still possible to use a single haplotype from an individual for interspecific comparisons. The haplotypes from an individual form a closely related lineage; therefore, the selection of ATRR haplotype should not affect the results of a phylogenetic or population genetic analysis. Specimens were obtained from Lago Rojo, Puerto Rico; Gainesville, FL; near the Great Smoky Mountains National Park in Tennessee; and Morgantown, WV. A description of DNA extraction and polymerase chain reaction covers the primers and amplification of the ATRR. Cloning and sequencing procedures are also described. 3 figures and 26 references
Main Term(s): Criminology; North America
Index Term(s): Death investigations; DNA fingerprinting; Investigative techniques; Time of death determination
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