Detection of single‐base mutation by affinity capillary electrophoresis using a DNA‐polyacrylamide conjugate

Abstract

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.

Quantitative SNP genotyping by affinity capillary electrophoresis using PEG‐oligodeoxyribonucleotide block copolymers with electroosmotic flow

“…Thus far, the SNP detection methods based on ACE have employed various separating media, such as hydrogels , nanoparticles , and polymers . In 2006, Krylov and coworkers reported an ACE using a MutS protein as an affinity probe, which can bind specifically to a single‐base‐mismatched site within dsDNA .…”

“…There are two methodologies for introducing a probe ODN into a CE system. First, the probe ODN is fixed in a stationary phase or a pseudostationary phase by covalently tethering to the inner surface of a capillary tube or a sieving matrix , respectively. Consequently, the probe ODN is prohibited from migrating during electrophoresis of the sample DNA.…”

“…Such chemical modification permits the probe ODN to migrate with an attenuated velocity compared to that of an unmodified ODN. These studies have successfully demonstrated that the target ssDNA is clearly separated from its single base substituted ssDNA (nontarget ssDNA), allowing for facile SNP detection .…”

Affinity capillary electrophoretic DNA separation using PEG‐oligodeoxyribonucleotide block copolymers: Relationship between peak resolution and affinity strength

“…Optimizing the various analytical conditions, we separated a mixture of chemically synthesized ssDNA and a single-base-substituted one. In addition, the practical effectiveness of this approach was demonstrated using PCR products as samples [20].…”

High‐Performance Sieving Electrophoresis for Single‐Nucleotide Polymorphisms with a Structuring Hydrogel Network

“…Furthermore, those methods involved cumbersome procedures such as denaturing, refolding, or reannealing of polymerase chain reaction products. Although affinity CE is suitable for detection of known mutation, it requires enormous efforts to optimize the separation condition . We demonstrated feasibility of Tetra‐PEG‐gel to detect known or unknown SNPs with conventional electrophoresis by injection of PCR products without any treatments.…”