Cationic comb-type copolymers for DNA analysis.
Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.
Kim
Won Jong
WJ
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan.
Sato
Yuichi
Y
Akaike
Toshihiro
T
Maruyama
Atsushi
A
D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics; D020137:Base Pair Mismatch; D032701:Biomimetics / Q000379:methods; D065731:Cytochrome P-450 CYP2C19; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics; D018832:Molecular Chaperones / Q000138:chemical synthesis / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods
D015342:DNA Probes; D018832:Molecular Chaperones; D009841:Oligonucleotides; D011108:Polymers; D004247:DNA; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19