Detection of DNA mutations by fluorescence resonance energy transfer-based preferential homoduplex formation assay.
Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET-PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET-PHFA detected KRAS mutations (1.25-50%) from all cell line DNA titration samples.
Kitano
Shiro
S
Life Science Research Laboratory, Toppan Printing, Kisarazu, Chiba 292-0818, Japan. shiro.kitano@toppan.co.jp
Nakayama
Mashimo
M
Yamane
Akio
A
Tsukahara
Yusuke
Y
Amano
Masahiko
M
D000818:Animals; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction; D018631:ras Proteins / Q000235:genetics
D018631:ras Proteins