Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts
| Autor: | Yongxi Zhao, Dexin Zhang, Qing Zhang, Chunhai Fan, Feng Chen, Xiaolei Zuo |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Telomerase Metal Nanoparticles 010402 general chemistry 01 natural sciences Biochemistry 03 medical and health sciences chemistry.chemical_compound Primer dimer Cell Line Tumor Humans Molecular Biology Helicase-dependent amplification Polymerase DNA Primers Enzyme Assays biology Organic Chemistry DNA Helicases Nucleic acid amplification technique Molecular biology 0104 chemical sciences 030104 developmental biology chemistry Cancer cell biology.protein Nucleic acid Molecular Medicine Gold Artifacts Dimerization Nucleic Acid Amplification Techniques DNA HeLa Cells |
| Zdroj: | Chembiochem : a European journal of chemical biology. 17(12) |
| ISSN: | 1439-7633 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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