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.

Keywords: KRAS mutations; ctDNA; enzymatic on/off switch; multiplex PCR; mutation detection.

Figure 1

The analysis of overall survival rate according to KRAS status in colorectal cancer patients. Colorectal cancer patients with KRAS mutations have significantly shorter survival time than patients with wild‐type KRAS. (P < .0001)

Figure 2

The assay establishment for the KRAS mutation detection mediated by an enzymatic on/off switch. A, An illustration showing the multiplex PCR experimental design by on/off switch. B, Taking the 12GCT and 13GAC of KRAS as examples, the mutant template and Wt template were diluted in 10‐fold serial and amplified by PCR with mutation‐specific primer. The length of the PCR products was both 155 bp. C, Establishment of a multiplex PCR system with codon 12, as the melt curve and amplification plot shows, the detection limit reach to 10² copies, and the specificity can reach to 10⁶

Figure 3

The current assay would not obtain false‐positive signals of KRAS gene mutations in pooled DNA sample of health control. As the top melt curve and amplification plot shows, the pooled DNA sample could be amplified by the wild‐type primers but not be amplified by the codon 12 mutation primers of KRAS. As the bottom melt curve and amplification plot shows, the DNA pool sample could be amplified by the KRAS wild‐type primers but could not be amplified by the codon 13 mutation primers of KRAS

Figure 4

The feasibility was tested for 7 hotspot mutations detecting of KRAS gene in cancer patients ctDNA samples. A, As the top melt curve and amplification plot shows, two of fourteen samples could be detected the codon 12 mutations of KRAS gene; as the bottom melt curve and amplification plot shows, one of fourteen samples could be detected the codon 13 mutation of KRAS gene. B, The sequencing of KRAS gene contained codon 12 and codon 13 of the three samples