Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis

Anal Chem. 2012 Jul 3;84(13):5645-52. doi: 10.1021/ac3008016. Epub 2012 Jun 20.

Abstract

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbocyanines / analysis
  • Colorectal Neoplasms / diagnosis*
  • Colorectal Neoplasms / genetics*
  • DNA / analysis
  • DNA / genetics*
  • Deoxyuracil Nucleotides / analysis
  • Feces / chemistry*
  • Humans
  • Multiplex Polymerase Chain Reaction / methods
  • Mutation Rate
  • Mutation*
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity

Substances

  • 3'-deoxy-5-(cyanine dye 3)uridine 5'-trisphosphate
  • Carbocyanines
  • Deoxyuracil Nucleotides
  • cyanine dye 5
  • DNA