Identification of base pairs in single-nucleotide polymorphisms by MutS protein-mediated capillary electrophoresis.
Single-nucleotide polymorphisms (SNPs) are widespread genomic variations, which are associated with serious health disorders and drug resistance. Multiple clinical applications and studies of global population genetics require fast and informative analysis of SNPs. Most of conventional methods sense the presence of the SNP but cannot identify the base pair in it. Here we report simple identification of base pairs in SNPs without DNA sequencing. Our approach is based on the unique ability of MutS protein to bind different single-nucleotide mismatches in DNA with different affinities. Conceptually, the DNA in question is mixed with reference DNA, melted, and reannealed. If the DNA in question has an SNP, the products of reannealing will have two different single-nucleotide mismatches, which provide a base-pair-specific signature of the SNP. The products of reannealing are mixed with MutS, equilibrated, and separated by equilibrium capillary electrophoresis of equilibrium mixtures with MutS in the run buffer. The pattern of migration times of DNAs with mismatches is used for unequivocal identification of the base pair in the SNP. In addition to its ability to identify base pairs in SNPs, the new analytical approach is fast, simple, highly sensitive, and requires no quantitation. It will find applications in studies of heterogeneity of base pairs in known SNPs in large human populations.
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Andrei P
AP
Drabovich
Sergey N
SN
Krylov
D020029:Base Pairing; D004247:DNA / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013824:Thermus / Q000737:chemistry; D013997:Time Factors
D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein