Microchip Electrophoresis of Tagged Probes Incorporated with One-Colored ddNTP for Analyzing Single-Nucleotide Polymorphisms

Abstract

We demonstrate a simple and rapid method for SNP typing, allele frequency determination, and trace mutant analysis that works with even an inexpensive detection system. This method is based on microchip electrophoresis of tagged probes incorporated with one-colored ddNTP (METPOC). The assay uses dye terminator incorporation into a pair of probes of different lengths specific to wild- and mutant-type targets, respectively. They are hybridized to the targets prior to ddNTP-Cy-5 incorporation, which occurs only for a matched probe-target duplex. Because the extension reactions for the two probes are carried out simultaneously in one tube and the products from both probes are analyzed in one channel by one-color fluorescence detection, an accurate comparative analysis of SNPs is possible. SNP typing as well as allele frequency determination in the range above 0.1% can easily be carried out using a commercial microchip electrophoresis system in a few minutes.

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

“…In our work the detection limit is as low as 0.01 pM. The sensitivity was comparable with or even exceeded the reported methods in which fluorescent (Twist et al, 2004), colorimetric electrophoretic (Li et al, 2003) or piezoelectric (Pang et al, 2006;Feng et al, 2007) approaches were used. Scheme 1 offered the fabrication and operation principle of the DNA biosensor.…”

Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

“…Quantifying allele frequency in DNA pools is important in association studies between SNPs and susceptibility to diseases [4]. To determine a mutant allele frequency using the proposed method, mutDNA and wtDNA were mixed at ratios of 0, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0.…”

“…Various heterogeneous and homogeneous methods for SNP detection have been reported. The heterogeneous formats for SNP detection generally require separation [3] or immobilization of the capture probe on a solid support phase such as a chip [4], a magnetic bead [5] or a nanoparticle [6]. This results in multiplex steps, high costs and longer analytical times.…”

High‐throughput mutational screening for beta‐thalassemia by single‐nucleotide extension

“…In a recent work, Li et al [17] used microchip electrophoresis and dye terminator incorporation into a pair of probes of different lengths specific to wild-type or mutant targets, respectively. The extensions of the probes are carried out simultaneously in one tube and the products, analyzed by one-color fluorescence detection, lead to SNP genotyping.…”