title: Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts.
abstract: Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts.
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: Department of Respiratory Medicine, The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xiwu Road, Xi'an, Shaanxi, 710049, P. R. China.
authors.forename: Dexin
authors.initials: D
authors.lastname: Zhang
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: Qing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: 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: Xiaolei
authors.initials: X
authors.lastname: Zuo
authors.affiliation: 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. yxzhao@mail.xjtu.edu.cn.
authors.forename: Yongxi
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D016477:Artifacts; D045744:Cell Line, Tumor; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism*; D019281:Dimerization; D057075:Enzyme Assays / Q000379:methods*; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D019098:Telomerase / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006046:Gold; D019098:Telomerase; D004265:DNA Helicases
keywords: helicase-dependent amplification; nanoparticles; polymerase chain reaction; primer-dimer artifacts; telomerase activity; zero-background