Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms.
DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3' ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.
Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, China, 421001.
Jia
J
Zhang
Kai
K
Li
Zemin
Z
Deng
Duanfang
D
Liao
Weiyi
W
Fang
Xu
X
Zhang
D001483:Base Sequence; D015246:Deoxyribonuclease EcoRI / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D009841:Oligonucleotides; D015246:Deoxyribonuclease EcoRI