dcDegenerate Oligonucleotide Primed-PCR for Multilocus, Genome-wide Analysis From Limited Quantities of DNA

• NCJ Number: 308561
• Author(s): Michelle D. Bonnette; Victoria R. Pavlova; Denise N. Rodier; Lindsay P. Thompson; Edward L. Boone; Kelly L. Brown; Kristin M. Meyer; Michelle B. Trevino; Jarrod R. Champagne; Tracey Dawson Cruz
• Date Published: September 2009
• Length: 11 pages
• Grant Number(s): 2005-DA-BX-K002
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Development and Evaluation of a Whole Genome Amplification Method for Accurate Multiplex STR Genotyping of Compromised Forensic Casework Samples

Annotation

This paper examines the impacts of altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases; it notes the research methodology, outcomes, and implications.

Abstract

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples. Experiments involved altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases. Increasing the degeneracy of the primer and the number of cycles that use a low annealing temperature should improve the nonspecific amplification of the DOP-PCR reaction. The addition of proofreading enzymes should allow for longer amplification products, increasing the genome coverage of the reaction. Low-input DNA quantities were examined for the primer and the cycle number studies using standard DOP-PCR parameters. The optimized DOP-PCR technique was then implemented for the polymerase study. All DOP-PCR products were amplified by using a multiplex microsatellite amplification kit to evaluate products from multiple chromosomes, followed by separation and detection by capillary electrophoresis. The 10 N primer, 12 nonspecific cycles, and the addition of the DeepVent proofreading enzyme all significantly increased the number of short tandem repeat alleles successfully amplified. All modifications also lowered the rate of allele drop-in, or sporadic additional allele occurrence, when compared with DOP-PCR results published earlier. Further, an average of >0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results showed that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples. (Published Abstract Provided)