Locked nucleic acid probe enhances Sanger sequencing sensitivity and improves diagnostic accuracy of high-resolution melting-based KRAS mutational analysis

Abstract

Background: Sanger sequencing is the gold standard for mutational analysis and widely used after high resolution melting (HRM) screening. However, the sensitivity of this method may be insufficient for identifying low frequency mutations. Therefore, for accurate diagnosis, enhanced sensitivity is warranted.

Methods: We designed a wild-type blocking cycle sequencing method using locked nucleic acid (LNA) probe (LNA-Sanger sequencing) for codons 12 and 13 of KRAS exon 2. We analyzed the sensitivities of HRM, conventional Sanger sequencing, and LNA-Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) reference standard samples with low frequency (5%) mutations in codons 12 and 13.

Results: Use of LNA probe significantly improved the sensitivity of Sanger sequencing (p=0.0003). Sensitivities of KRAS mutation tests were as follows: HRM, 5%; conventional Sanger sequencing, 10%; and LNA-Sanger sequencing, 5%. FFPE samples with 5% mutation were accurately diagnosed by LNA-Sanger sequencing, whereas it was difficult to identify the mutations by conventional Sanger sequencing.

Conclusions: LNA-Sanger sequencing is a facile technique for the enrichment of mutant alleles and useful for the accurate diagnosis of HRM-positive cases with low frequency mutations.

Keywords: High resolution melting; KRAS codons 12 and 13; Locked nucleic acid; Mutational analysis; Sanger sequencing.