Detection of Low-Abundance KRAS Mutations in Colorectal Cancer Using Microfluidic Capillary Electrophoresis-Based Restriction Fragment Length Polymorphism Method with Optimized Assay Conditions

Abstract

Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient’s age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.

A non-invasive genomic diagnostic method for bladder cancer using size-based filtration and microchip electrophoresis

“…The MCE system had a higher sensitivity, over 10-fold higher than that of the PAGE assay, as indicated in Fig. 5C-E. Like others have confirmed previously [47], the high sensitivity of the MCE system can reduce false-negative results in cancer diagnosis. Moreover, execution time and reagent consumption were significantly decreased compared to traditional analytic methods for disease monitoring.…”

Association of a let-7 KRAS rs712 polymorphism with the risk of breast cancer

“…Previous studies have shown that mutations in the KRAS gene, which leads to a change in KRAS sensitivity or elevated KRAS expression, are correlated with the development of various human cancers (van Grieken et al, 2013;Zhang et al, 2013). miRNA are trans-acting factors that function as regulators of cancer development, progression, and metastasis by binding to specific target genes, including KRAS.…”

Advances in Microfluidics and Lab-on-a-Chip Technologies

“…As an example of what has been accomplished recently, micro CE integrated systems have been used for quantitative detection of low-abundance mutations of the KRAS gene from paraffin tissue sections of colorectal cancer. These systems have nano-liter sample introduction components leading to CE separation of the target genes and detection by laser-induced fluorescence (LIF); all being accomplished in minutes or even seconds (Zhang, et al, 2013;Xu, et al, 2010). DNA purification He and co-workers used capillary zone electrophoresis for purification of sequencing fragments (He, et al, 2000).…”