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

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 (<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.

DNA Methylation-regulated miR-193a-3p Dictates Resistance of Hepatocellular Carcinoma to 5-Fluorouracil via Repression of SRSF2 Expression

“…Therefore, the genomic profiling at both DNA methylation and miR expression levels was carried out in both QGY-7703 and SMMC-7721 for the DNA methylation regulated miRs having a key role in the 5-FU resistance of HCC cells. Among 39 differentially expressed miRs that were identified by a Solexa sequencing-miRomic analysis, 4 miR-193a-3p was one of the most differentially expressed between SMMC-7721 and QGY-7703: 11.21-fold higher in former than in the latter by Solexa sequencing (Fig. 1D) and 4.44-fold higher by qRT-PCR-based validation (Fig.…”

“…Among the epigenetic entities, which refer to the DNA sequence-independent mechanisms underlying the cross-cell generational transmission of gene expression memory, DNA methylation (the addition of the methyl group at the cytosine ring of the 5Ј-CpG-3Ј sequence) is the best characterized and is regarded as a promising molecular indicator for the existence and/or prognostic state of cancer (4,5). Micro-RNAs (miRs) 3 are small noncoding RNAs that regulate the expression of the protein-coding genes, including the genes controlling the DNA methylation state of the genome (6), at both RNA stability and translational levels in a sequencing-specific manner.…”

“…We also established the methylomic profiles of both cell lines by probing a customized Nimblegene 0.38 million oligonucleotide CpG island array with the heavily methylated DNA fraction from a methylated DNA-binding domain-based capture procedure (28). The miR-193a gene was at the top of the most differentially methylated CpG islands (Ͼ3000) 4 : hypermethylated in QGY-7703 and hypomethylated in SMMC-7721 (methylation index, ϩ1.22 versus Ϫ2.35; Fig. 1F), negatively correlating with the miR-193a-3p expression.…”

Whole genome amplification in preimplantation genetic diagnosis

“…Recently, a modification of the traditional DOP-PCR reaction (dcDOP-PCR), including the use of a more degenerate primer (10 nucleotides) and 12 nonspecific cycles, was developed by Bonnette et al (2009). In this protocol, the longer amplification products were generated by additional proofreading enzymes; thus, increasing the genome coverage of the reaction.…”

Multiplex Short Tandem Repeat Amplification of Low Template DNA Samples with the Addition of Proofreading Enzymes*

“…Typically, the addition of an enzyme that has proofreading capability results in longer fragments, although the exonuclease activity reduces the overall processivity of the reaction (9,10). Previous studies have shown that combining these proofreading enzymes with Taq polymerase for preamplification is the best approach for increasing fragment length and improving genome coverage, without compromising the speed of the reaction (6,7).…”