dcDegenerate oligonucleotide primed-PCR for multilocus, genome-wide analysis from limited quantities of DNA.
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 (<or= 0.100 ng). 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 show 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.
Bonnette
Michelle D
MD
Texas Department of Public Safety, Crime Laboratory Service, TX, USA.
Pavlova
Victoria R
VR
Rodier
Denise N
DN
Thompson
Lindsay P
LP
Boone
Edward L
EL
Brown
Kelly L
KL
Meyer
Kristin M
KM
Trevino
Michelle B
MB
Champagne
Jarrod R
JR
Cruz
Tracey Dawson
TD
D002875:Chromosomes; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D019075:Electrophoresis, Capillary; D016678:Genome; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004247:DNA