Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.
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.
van Eijk
Ronald
R
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
van Puijenbroek
Marjo
M
Chhatta
Amiet R
AR
Gupta
Nisha
N
Vossen
Rolf H A M
RH
Lips
Esther H
EH
Cleton-Jansen
Anne-Marie
AM
Morreau
Hans
H
van Wezel
Tom
T
D000230:Adenocarcinoma / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005557:Formaldehyde; D005820:Genetic Testing / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins