Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

J Vis Exp. 2015 Oct 8;(104):53190. doi: 10.3791/53190.

Abstract

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Cell Line
  • DNA / genetics*
  • DNA / isolation & purification*
  • DNA Mutational Analysis / methods*
  • Formaldehyde / chemistry
  • Humans
  • Mutation
  • Paraffin Embedding
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins B-raf / genetics*
  • Reference Standards

Substances

  • Formaldehyde
  • DNA
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf