Type it hrm master mix

Genomic DNA was extracted using salting out method from venous blood samples. DNA quantification and qualification were estimated using Spectrophotometer (GE-NanoVue). To cover all exons of BRCA1 gene, 41 primers were used according to previous study (Ava Kwong et al., 2012) for HRM mutation screening. DNA amplification was performed by 36 plates RotorGeneQ 5Plex HRM (Qiagen, California, USA). RotorGene Q 5Plex HRM (Qiagen, California, USA) was used for PCR-HRM amplification. Each reaction was performed in a final volume of 20 μl containing 10 μl Type-It HRM Master Mix (Qiagen, California, USA), 4 μl primer (5 pM forward and reverse primer), 2 μl DNA 10 ng and H2O up to 20 μl. The PCR profile was performed as follows: pre-activation at 950C for 5 minute, followed by 40 cycles of denaturation at 950C for 10 seconds, 30 seconds annealing at 54-620C and 10 seconds extension at 720C. HRM amplification were done at 60-980C with 0.10C temperature increment. Melting curve was analyzed by Rotor Gene -Pure Detection version 2.1.0 (build 9) (Qiagen, California, USA) software. Afterwards, aberrant patterns were sequenced using Sanger sequencing method.

Using the gene-specific primers KRAS forward [5′-AGAGGCCTGCTGAAAATGACTGAA-3′] and KRASreverse biotin [5′-TTAGCTGTATCGTCAAGGCACTCT-3′], PCR was performed on 5-10 ng/µL template DNA with a Type-it HRM Mastermix (Qiagen). The cycling profile comprised an activation step at 95°C for 5 min, followed by 40 cycles of amplification, including 30 s at 95°C, 20 s at 58°C, and 30 s at 72°C on a Rotor-Gene Q cycler (Qiagen). A melt analysis was performed after the PCR as a quality control. PCR products were purified via Sepharose beads on the PyroMark Q24 Working Station (Qiagen) and sequenced in the PyroMark Q24 using the sequencing primer KRAS seq_forward [5′- . The default sequence to analyze was set to [5′-G/NGTGGCGTAG-3′], with the dispensation order of [5′-ACTGACTACGATCGTA-3′] enabling a later transversion to [5′-GG/NTGGCGTAG-3′], [5′-GGTG/NGCGTAG-3′], or [5′-GGTGG/NCGTAG-3′] to analyze every common mutation occurring in codons 12 and 13 of the KRAS gene.