Abstract 4855: Use of blocker locked nucleic acid (LNA™) oligonucleotides in cycle sequencing for improved limits of detection (BLOC-Sequencing)

Epidermal growth factor receptor (EGFR) antagonists, such as cetuximab and panitumumab, are therapeutic agents that can be effective in colorectal cancer (CRC) treatment. It has been shown that 40% of CRC tumors have activating K-RAS exon 2 codon 12 and 13 mutations and that these mutations may be associated with a poor response to EGFR antagonists. Very high sensitivity detection of such diagnostic biomarkers is necessary to determine the presence or emergence of drug resistant tumor cell populations. The use of Locked Nucleic Acid (LNA™) containing oligonucleotides (oligos) have been used in microRNA (sample preparation), RNA (in situ hybridization), and DNA (SNP detection using allele specific PCR) applications. The advantage of using LNA-containing oligos is that the denaturation temperature of LNA-oligo:DNA duplex is increased as compared to the DNA:DNA duplex To further the limit of detection of Sanger sequencing, a LNA-based approach has been developed to selectively block the sequencing of the wild-type DNA and allow the sequencing of DNA containing any mutation (BLOC-Sequencing). During cycle sequencing, an additional annealing step is added to hybridize the LNA containing oligo to the template DNA. Then, a denaturing step is performed at a temperature at which the LNA-containing oligo remains annealed to the wild-type sequence; however, the LNA oligo denatures from the mutant sequence. The sequencing primer then anneals to the mutant sequence and subsequently extended. Since LNA-containing oligos block amplification of the wild-type sequence, the mutant-containing DNA will be sequenced in a non-biased manner. Preliminary results using two sequences in a 1:1 ratio differing in only 2 nucleotides within the range where the LNA blocking oligo anneals demonstrated complete blocking of one sequence (sequence 1) where the LNA containing oligo was 100% complementary. The unblocked sequence (sequence 2) sequenced successfully, with minimal background due to sequence 1. Straight Sanger sequencing results of the 1:1 mixture without the LNA blocking oligo gave equal proportion of the two sequences present. To show applicability for use of this methodology in CRC samples, the limit of detection for multiple codon 12 and codon 13 K-RAS exon 2 mutations with and without the addition of the K-RAS exon 2 wild type specific LNA oligo in both the forward and reverse directions will be demonstrated. In addition, we will compare the results of 30 previously genotype