Internal Validation of Human Mitochondrial DNA Quantification Using Real-Time PCR

• NCJ Number: 246021
• Author(s): Marc L. Sprouse B.S., Nicole R. Phillips Ph.D., Mark F. Kavlick B.S., Rhonda K. Roby
• Date Published: July 2014
• Length: 7 pages
• Agencies: NIJ-Sponsored
• Award: Development of an Expert System for Automated Forensic mtDNA Data Analysis; FY 2008 Forensic DNA Unit Efficiency Improvement - University of North Texas Center for Human Identification Laboratory for Molecular Identification

Annotation

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data.

Abstract

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real-time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/ìL or approximately six human mtDNA copies/ìL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re-purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing. Abstract published by arrangement with Wiley.