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

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

Author information

1
Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University.
2
ABION CRO.
3
ABION Inc., R&D Center.
4
The Center for Anti-Cancer CDx, N-Bio, Seoul National University.
5
Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University; The Center for Anti-Cancer CDx, N-Bio, Seoul National University; ykeeshin@snu.ac.kr.

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.

PMID:
26484710
PMCID:
PMC4692650
DOI:
10.3791/53190
[Indexed for MEDLINE]
Free PMC Article

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