Hotstarttaq plus dna polymerase

The genomic DNA for each EOC cell line (OV90, TOV21G, TOV112D, TOV81D, TOV1946, OV1946 and TOV2223) was amplified for each of the five exons of IGFBP7 and flanking introns using previously published primer sets [54 (link)] (Additional file 1). PCR reactions were performed as described above, but using the following PCR thermal cycler conditions for each primer set: 95°C for 3 minutes, 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes for 35 cycles. QIAGEN HotStart Taq Plus DNA Polymerase and 5X Q-solution (Cat. No. 203603, QIAGEN Inc. Mississauga, ON, Canada) were used for the amplification of the G-C rich exon 1. PCR products were then subjected to a Sanger sequencing protocol using 3730XL DNA Analyzer systems from Applied Biosystems at the McGill University and Genome Québec Innovation Centre (gqinnovationcenter.com). Sequencing chromatograms were analysed using 4Peaks Version 1.7.2. Sequence alignment was performed using the ClustalW multiple sequence alignment platform from the European Bioinformatics Institute [55 (link)] and compared with NM_001553 (IGFBP7) sequence. Variants identified were compared with those reported in the Single Nucleotide Polymorphism (dbSNP) database (http://www.ncbi.nlm.nih.gov/projects/SNP/).

The sodium bisulfite treatment method was used to assess methylation of IGFBP7 using the QIAGEN EpiTect Bisulfite Kit (QIAGEN Inc., Mississauga, ON, Canada) according to the manufacturer’s instructions. Two μg of genomic DNA from each EOC cell line was used for the conversion. Methylation-specific PCR was carried out using previously published primers (Additional file 1) [37 (link)]. PCR amplification of DNA was performed using QIAGEN HotStart Taq Plus DNA Polymerase (QIAGEN Inc. Mississauga, ON, Canada) as described above under the following PCR conditions: with the methylated primers for 35 cycles at 95°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes; and with the unmethylated primers for 35 cycles at 95°C for 5 minutes, 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The PCR products were resolved in 1% gel electrophoresis.