Hot start taq enzyme

For the polymorphism analysis, DNA was extracted using the QIAamp Blood DNA Mini-Kit (Qiagen) according to the manufacturer's instructions. Genotyping was performed through PCR amplification using the Hot Start Taq enzyme (Qiagen). Following amplification, the products were purified with the ExoSap enzyme (USB products) and sequenced bidirectionally using the BigDye Terminator v3.1 Kit (Applied Biosystems, USA). Electrophoresis was run in the automated sequencer model 3500 (Applied Biosystems, USA).
For the analysis of mutations in the BRCA1 and BRCA2 genes and the subsequent separation of the participants into the three study groups, a multiplex PCR amplification of all coding exons of the BRCA1 (NCBI; NM_007294.3) and BRCA2 (NCBI; NM_000059.3) genes and their respective flanking intronic regions was performed, followed by bidirectional sequencing using two platforms (ABI 3500 XL sequencer) and a new generation sequencer (Ion Torrent PGM, Applied Biosystems). In addition, large rearrangements were investigated using the multiplex ligation-dependent probe amplification (MLPA) technique.

Genomic DNA from 5-aza-dC–treated or mock-treated cells was isolated via PureLink Genomic DNA Purification Kit (Invitrogen) per manufacturer's protocol. Subsequently, genomic DNA aliquots were treated with sodium bisulfite via EZ DNA Methylation-Gold Kit (Zymo Research), followed by PCR to amplify the PDLIM2 promoter by Hot-Start Taq enzyme (Qiagen). Primers were designed to recognize the bisulfite-modified regions (−1084 to −800) of the PDLIM2 promoter (forward 5′-AGAGGAGTTTATATATATTTAGG, reverse 5′-TACCTAACAACCCTCTCTCC). The PCR products were then directly employed for DNA sequencing or subcloned into the SmaI restriction site of pEGFP-N2 (Clontech) for single colony sequencing to determine the methylation status of the CpG dinucleotides within the PDLIM2 promoter.

Genomic DNA from 5-aza-dC-treated or DMSO mock-treated cells were isolated using the PureLink Genomic DNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Genomic DNA aliquots were then treated with sodium bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA), followed by PCR to amplify the pdlim2 promoter using Hot-Start Taq enzyme (Qiagen, Hilden, Germany). The PCR products were used for DNA sequencing to determine the methylation status of the CpG dinucleotides within the pdlim2 promoter.