NCJ Number
228279
Date Published
2009
Length
115 pages
Annotation
In order to increase the information provided by mitochondrial DNA (mtDNA) analysis by targeting additional sequence polymorphisms outside the HVI/II regions, the project described in this report developed a highly sensitive, easy-to-use, 5-plex and 10-plex PCR and linear array assay for simultaneous analysis of polymorphic regions in the noncoding and coding regions.
Abstract
This assay targets 61 polymorphic sites distributed throughout the mitochondrial genome, using 15 primer pairs and 105 sequence-specific oligonucleotide probes immobilized in lines on a nylon membrane. The 5-plex PCR is used to amplify 5 regions that range in size from 314-444 bp; the 10-plex PCR is used to amplify 10 regions that range in size from 103-183 bp. The 5-plex probe panel consists of 59 probes and targets variation at 14 HVI sites, 11 HVII sites, 8 coding region (CR) sites, and 2 variable region I (VRI) sites. The 10-plex probe panel consists of 46 probes and targets 17 CR sites, 4 VRI sites, and five VRII sites. A population study was conducted in order to determine the power of discrimination for the new expanded HV+ array. In order to determine the assay limitations, a developmental validation study was conducted. A sensitivity study showed that both the 5-plex and 10-plex assays were highly sensitive assays, with the 10-plex being more sensitive. Additional developmental studies - including reproducibility, precision and accuracy, and environmental samples - were also completed. In addition, a procedure was validated for automating the typing assay, and scanning and interpretation software were modified for use with the 5-plex and 10-plex assays. Up to 48 samples can be typed manually or automated in less than 2 hours. The data can now be quickly analyzed with the Strip Scan Mitotyper software. 19 figures, 6 tables, and 42 references
Date Published: January 1, 2009