Table 1.
K-ras codon 12 mutation rate and clinicopathological parameters.
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.
(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.
Table 2.
Characteristics of the K-ras codon 12 in different colon cancer cells confirmed by sequencing analysis.