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Real-Time Polymerase Chain Reaction Quantification of Canine DNA

NCJ Number
217223
Author(s)
Jeffrey J. Evans B.S., Elizabeth J. Wictum B.S., Cecilia T. Penedo Ph.D., Sreetharan Kanthaswamy Ph.D.
Date Published
January 2007
Length
4 pages
Annotation
This study evaluated two quantitative polymerase chain reaction (QPCR) assays for detecting and quantifying forensic canine DNA: the canine Melanocortin-I Receptor (MCIR) gene and a canine short interspersed element (SINE) reported by Walker et al.
Abstract
Both the MCIR TaqMan assay and the SINE SYBR Green I assay were found to be effective tools for the quantification of dog DNA. Both are canid-specific and sensitive to approximately one cell's DNA mass. The researchers selected the MCIR TaqMan assay over the SINE SYBR Green I assay for use in forensic casework because of the shorter run time, the toxicity of SYBR Green I, and the belief that the diploid copy might be more predictive of short tandem repeat (STR) genotyping success than the multiple and variable copy numbers of SINEs. The two primary limiting factors regarding the accuracy and reproducibility of QPCR assays are the accuracy of pipetting and the quality of the standard curve dilutions. In order to counter potential pipetting discrepancies, the template volume should be increased to more than 1 ml. Also, the use of a robotic system of PCR setup would improve assay precision. As the target DNA concentration is measured relative to a standard, the standard must be accurately quantified. A stable, calibrated reference standard will enhance the accuracy of these assays. The criteria for selecting a QPCR target region were that the sequence be conserved within a species and between closely related species, that it contain sufficient sequence variation between taxonomic families to exclude nontarget DNA, and that sequence data be readily available for a variety of species. The description of the methods used addresses primer and probe design, thermal cycling parameters, and the establishment of a standard curve. 2 tables, 2 figures, and 19 references

Date Published: January 1, 2007