Use of BLOCker sequencing (blocking oligonucleotide cycle sequencing) after ice COLD-PCR for detection of K-RAS mutations.

Abstract

e13547 Background: The epidermal growth factor receptor (EGFR) antagonists, cetuximab and panitumumab, can be effective in colorectal cancer treatment. However, activating K-RAS mutations in codons 12 and 13 are associated with a poor EGFR antagonist response. As these mutations are seen in ~40% of CRC, high sensitivity assays for detection of these mutations will aid in patient treatment and disease monitoring. Methods: Ice COLD-PCR (Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) uses a reference sequence oligonucleotide (RS-oligo) to enhance detection of all low level mutations during PCR. The RS-oligo is complementary to one wild-type strand, so only one wild-type strand but both mutant DNA strands are amplified. This results in linear WT sequence amplification but exponential mutant sequence amplification. Ice COLD-PCR methods used in these studies can detect 0.05 – 0.01% K-RAS mutant DNA. For low level mutation confirmation we added BLOCker-oligos to sequencing reactions to block wild-type and allow mutant DNA sequencing (BLOCker-Sequencing). For blocking to occur, additional hybridization and denaturing steps are added to the cycle sequencing program. This insures that the BLOCker-oligo:WT DNA remains intact but the BLOCker-oligo:Mutant DNA is denatured. A sequencing primer overlapping the BLOCker-oligo 5’ end anneals to the mutant strand that is subsequently extended. Results: BLOCker sequencing was used to confirm presence or absence of low level K-RAS mutations after Ice COLD-PCR. When a K-RAS mutation is present at 0.05%-0.01%, the mutant peak may appear as baseline noise. When BLOCker Sequencing of these samples is performed, the presence of a mutation at this low level is enhanced in the BLOCker Sequencing electropherogram. There is no enhancement if the sample does not contain a mutation. Conclusions: Following Ice COLD-PCR amplification, BLOCker sequencing was able to confirm the presence or absence of very low levels of K-RAS mutations in the starting samples. BLOCker Sequencing is a novel method to confirm low level mutation presence when there is a uncertain DNA sequencing electropherogram result following Sanger sequencing.