Usb exosapit

Following NGS sequencing, EGFR and KRAS (including codons 12, 13) mutations in samples from the RCRC set as well as KRAS mutations from RSCRR set were validated either by Sanger sequencing or Real-Time PCR. Samples from RSCRR set were pre-screened for EGFR mutations and, thus, not required further validation with orthogonal methods. EGFR mutations (including exons 18–21) were examined by Sanger Sequencing. Primer pairs were 5′-CTGAGGTGACCCTTGTCTCTG-3′ and 5′-CCAAACACTCAGTGAAAC-3′, 5′-TGCCAGTTAACGTCTTCCTT-3′ and 5′-CAGGGTCTAGAGCAGAGCAG-3′, 5′-CATTCATGCGTCTTCACCTG-3′ and 5′-TTATCTCCCCTCCCCGTATC-3′, and 5′-TGATCTGTCCCTCACAGCAG-3′ and 5′-GGCTGACCTAAAGCCACCTC-3′ for exons 18, 19, 20 and 21 respectively. Thermal cycling conditions included 5 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min and one cycle of 72 °C for 7 min. The PCR products were further purified with USB ExoSapit (GE Healthcare, Uppsala, Sweden) followed by cycle sequencing with the BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s protocol and resolved on an ABI 3500xL sequencer (Applied Biosystems). Sequence chromatograms were analyzed by Sequencher software (Gene Codes Corp, Ann Arbor, MI), followed by manual review. KRAS codon 12, 13 mutations were examined by pyrosequencing as described previously [33 (link)].