An enzymatic on/off switch-mediated assay for KRAS hotspot point mutation detection of circulating tumor DNA.

ABSTRACT

BACKGROUND: To detect the mutations of KRAS gene in colorectal cancer patients and other cancer patients, it is of value to develop non-invasive, sensitive, specific, easy, and low-cost assays. METHODS: Templates harboring hotspot mutations of the KRAS gene were constructed, and primers were designed for evaluation of the specificity, and sensitivity of detection system consisted of exonuclease polymerase-mediated on/off switch; then, gel electrophoresis and real-time PCR were performed for verification. The assay was verified by testing the DNA pool of normal controls and circulating DNA (ctDNA) samples from 14 tumor patients, as compared to Sanger sequencing. RESULTS: A specific and sensitive assay consisted of exonuclease polymerase-mediated on/off switch, and multiplex real-time PCR method has been established. This assay could detect <100 copies of KRAS mutation in more than 10 million copies of wild-type KRAS gene fragments. This assay was applied to test KRAS gene mutations in three cases of fourteen ctDNA samples, and the results were consistent with Sanger sequencing. However, this PCR-based assay was more sensitive and easier to be interpreted. CONCLUSION: This assay can detect the presence of KRAS hotspot mutations in clinical circulating tumor DNA samples. The assay has a potential to be used in early diagnosis of colorectal cancer as well as other types of cancer.