Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. Academic Article uri icon

Overview

abstract

  • Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

publication date

  • August 1, 2005

Research

keywords

  • Colonic Neoplasms
  • DNA, Neoplasm
  • Genes, ras
  • Mutation
  • Polymerase Chain Reaction

Identity

PubMed Central ID

  • PMC1867544

Scopus Document Identifier

  • 23844497341

Digital Object Identifier (DOI)

  • 10.1016/S1525-1578(10)60571-5

PubMed ID

  • 16049314

Additional Document Info

volume

  • 7

issue

  • 3