Heteroplasmies Detected in an Amplified Mitochondrial DNA Control Region From a Small Amount of Template

• NCJ Number: 222340
• Journal: Journal of Forensic Sciences Volume: 53 Issue: 2 Dated: March 2008 Pages: 306-311
• Author(s): Hiroaki Nakahara; Kazumasa Sekiguchi Ph.D.; Kazuhiko Imaizumi Ph.D.; Natsuko Mizuno; Kentaro Kasai Ph.D.
• Date Published: March 2008
• Length: 6 pages
• Publication Format: Article
• Publication Type: Report (Study/Research)
• Language: English
• Country: United States of America

Annotation

Since mitochondrial DNA (mtDNA) heteroplasmies (a state in which two or more haplotypes of mtDNA coexist in a single individual) often confound forensic identification, especially if they result from poor biological sampling, this study determined the ratio of heteroplasmy in samples that were amplified from a small amount of template mtDNA or a few cells, using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis.

Abstract

Findings show that more than half of the detected sequences (17 of 20, 15 of 20, and 14 of 20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. In addition, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These findings show the risk associated with using highly sensitive mtDNA techniques in forensic investigations due to the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample. In many cases, heteroplasmy can lead to confusion when the sequences are compared; for example, in some cases of maternal-individual identification, the ratio of heteroplasmy is sometimes significantly different between generations, and the haplotypes of each sequence are apparently mismatched. Consequently, this report advises that in analyzing mtDNA for identification, forensic scientists should recognize the potential risks, in that different haplotype of mtDNA sequences derived from potential heteroplasmy or from polymerase misincorporation can be potential problems when a very small amount of template DNA is amplified using high-sensitivity PCR. The description of materials and methods addresses DNA extraction from bloodstain samples, quantification of mtDNA, WBC samples, nested PCR, sequencing analysis, the measurement of proportion of each heteroplasmy, and the screening of heteroplasmy. 2 tables, 3 figures, and 25 references