title: Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.
abstract: Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine.
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University , Xianning West Road, Xi'an, Shaanxi 710049, P. R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University , Xianning West Road, Xi'an, Shaanxi 710049, P. R. China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences , Yuquan Road, Shanghai 201800, P. R. China.
authors.forename: Chunhai
authors.initials: C
authors.lastname: Fan
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University , Xianning West Road, Xi'an, Shaanxi 710049, P. R. China.
authors.forename: Yongxi
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D020137:Base Pair Mismatch; D065731:Cytochrome P-450 CYP2C19 / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment
chemical_list: D006046:Gold; D065731:Cytochrome P-450 CYP2C19; D004259:DNA-Directed DNA Polymerase