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.

Figure 1. Schematic view of microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform.

(a) Mismatched primer PCR. A KRAS gene fragment containing codon 12 was amplified from gDNA with mismatched primer, by which a base substitution was introduced to the amplicon and a Mva I restriction endonuclease recognition site was created for wild-type codon 12. (b) Mva I digestion. The amplicon from wild-type template could be cleaved into two fragments, the amplicon from mutant template could not be digested due to the loss of recognition site, and the amplicon from heterozygous template was halfly digested. (c) µCE. The digested amplicon was loaded into microfluidic chip and separated by CE according to the fragment length. The wild type template resolved into two peaks, the mutant template only showed one peak, and the heterozygous template resolved into three peaks. gDNA, genomic DNA. SR: sample reservoir; BR: buffer reservoir; SW: sample waste reservoir; BW: buffer waste reservoir. →, the direction of fluid flow during sample loading and separation modes.

Figure 2. Optimization of separation parameters for the microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform.

(A) The effect of field strength. Conditions for separation were 2% HPC, 3×TBE with varied field strength. (B) The effect of polymer concentration. Conditions for separation were 3×TBE, 180 V/cm with varied polymer concentrations. (C) The effect of buffer concentration. Conditions for separation were 2% HPC, 180 V/cm with varied ionic strength. (D) µCE-based RFLP analysis of the products of enzymatic digestion. Products were separated in sieving buffers with 3×TBE, 2×TBE, or 1×TBE. Separations were performed using 2% HPC under a 180 V/cm electric field.

Sensitivity analysis of the µCE-based RFLP platform.

SW480 cells carrying mutant KRAS were mixed with KRAS wild-type HT29 cells at various ratios and detected by Natural PAGE (A), direct sequencing (B), and µCE-based RFLP (C), respectively. Conditions for separation were 2% HPC, 3×TBE, and 180 V/cm.

Analysis of reproducibility.

(A) intra-assay. (B) inter-assay.

Figure 5. Electropherograms of the KRAS gene in six CRC cell lines.

Figure 6. Three representative KRAS gene mutation-positive CRC PETs detected by µCE-based RFLP but not by direct sequencing.

The empty arrows indicate the peaks of mutant fragments. The underlined bases in the sequencing results are codons 12 (exon 2) of KRAS. Mutant sites are marked with black arrows.