Assays

 

An important issue in KRAS genotyping is the method used for testing. Two basic methods have predominated in KRAS testing: dideoxysequencing and allele specific PCR. Sequencing has long been the most important method for detecting point mutations. The major disadvantage is that sequencing is not very sensitive, and especially in samples with low tumour content, analysis might be difficult.  Allele specific PCR is more sensitive but only tests for a subset of the most common mutations, whereas sequencing can detect all possible mutations. Recently, more specific methods have been developed for mutation testing and specific kits for KRAS testing are available. In the datasheet of the KRAS EQA scheme we ask about the method used for KRAS testing. Over time, as more data become available, this scheme will be able to provide more detailed information about how different methods are performing.

 

Methods for mutation analysis in general:

• Genotypic selection methods for the direct analysis of point mutations
  Parsons BL, Heflich RH , Mutation Research, 1997, 387:97-121

Methods for KRAS testing:

• High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies
  Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A , BMC Cancer, 2008, 8(142):1-14
• FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations
  Amicarelli G, Shehi E, Makrigiorgos GM, Adlerstein D , Nucleic Acids Research, 2007, 35(9):1-8
• Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing
  Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, Mino-Kenudson M, Lauwers GY, Loda M, Fuchs CS , Journal of Molecular Diagnostics, 2005, 7(3):413-421
• Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process
  Tatsumi K, Mitani Y, Watanabe J, Takakura H, Hoshi K, Kawai Y, Kikuchi T, Kogo Y, Oguchi-Katayama A, Tomaru Y, Kanamori H, Baba M, Ishidao T, Usui K, Itoh M, Cizdziel PE, Lezhava A, Ueda M, Ichikawa Y, Endo I, Togo S, Shimada H, Hayashizaki Y , Journal of Molecular Diagnostics, 2008, 10(6):520-526
• Rapid and large-scale method to detect K-ras gene mutations in tumor samples
  Lopez-Crapez E, Chypre C, Saavedra J, Marchand J, Grenier J , Clinical Chemistry, 1997, 43(6):936-942

Commercial kits:

• A commercial real time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations
  Angulo B, García-García E, Martínez R, Suárez-Gauthier A, Conde E, Hidalgo M, López-Ríos F , Journal of Molecular Diagnostics, 2010, 12(3):292-299

Last changed: 2011-11-21