Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.
J Mol Diagn
; 12(1): 27-34, 2010 Jan.
Article
en En
| MEDLINE
| ID: mdl-19959798
Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Pruebas Genéticas
/
Proteínas Proto-Oncogénicas
/
Proteínas ras
/
Mutación
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Guideline
Límite:
Humans
Idioma:
En
Revista:
J Mol Diagn
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2010
Tipo del documento:
Article
País de afiliación:
Países Bajos
Pais de publicación:
Estados Unidos