title: 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.
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan. Electronic address: ishige-t@chiba-u.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ishige
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan.
authors.forename: Sakae
authors.initials: S
authors.lastname: Itoga
authors.affiliation: Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Clinical Genetics, Chiba University Hospital, Chiba, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsushita
authors.affiliation: Division of Clinical Genetics, Chiba University Hospital, Chiba, Japan; Division of Clinical Mass Spectrometry, Chiba University Hospital, Chiba, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
mesh_terms: D004252:DNA Mutational Analysis; D011905:Genes, ras; D006801:Humans; D015335:Molecular Probes; D009841:Oligonucleotides / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D015335:Molecular Probes; D009841:Oligonucleotides; C477371:locked nucleic acid
keywords: High resolution melting; KRAS codons 12 and 13; Locked nucleic acid; Mutational analysis; Sanger sequencing