Mini-Midi-Mito: Adapting the Amplification and Sequencing Strategy of mtDNA to the Degradation State of Crime Scene Samples

• NCJ Number: 227234
• Journal: Forensic Science International: Genetics Volume: 3 Issue: 3 Dated: June 2009 Pages: 149-153
• Author(s): Cordula Berger; Walther Parson
• Date Published: June 2009
• Length: 5 pages
• Publication Format: Article
• Publication Type: Report (Study/Research)
• Language: English
• Country: United States of America

Annotation

For moderately degraded biological samples, which are encountered frequently in typical mitochondrial DNA (mtDNA) casework material, the authors developed 2 new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded control region (CR) consensus sequence.

Abstract

The development of four multiplex assays, the two "mini-multiplexes" and the two "midi-multiplexes" described, increased the success rate for the analysis of the entire control region sequences in highly and moderately degraded samples compared to conventional approaches traditionally used for forensic applications (HVS-1/HVS-II). The authors found that these multiplex assays met the demands of routine forensic mtDNA work that dealt with various kinds of biological samples which exhibited varying mtDNA degradation states. Handling the combined application of degradation-sensitive real-time quantitative PCR and the appropriate mtDNA amplification in actual casework samples over time yielded limits of detection that were 300-400 genome equivalents of target mtDNA for the midi-amplicons and 200-300 genome equivalents for the mini-amplicons. The upstream quantitation assay provided useful information for the selection of the appropriate amplification and sequencing strategy. The proposed procedure used a preceding mtDNA quantitation step by real time PCR with two different target fragments (143 and 283 bp) that approximately corresponded to the average fragment sizes of the different multiplex approaches in order to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes regarding quality of the results and required costs. Nine evidentiary hair shafts, all between 1.5 and 5 cm in length were subjected to mtDNA analyses. The hair shafts that were longer than 1.5 cm were cut into two equal pieces for independent analysis. The extraction process is described in detail, and an actual case work example that used the proposed method is presented. 3 figures, 3 tables, and 6 references