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Issue 38, 2015
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Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

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Abstract

Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.

Graphical abstract: Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

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Supplementary files

Article information


Submitted
03 Jun 2015
Accepted
11 Aug 2015
First published
25 Aug 2015

Nanoscale, 2015,7, 15852-15862
Article type
Paper

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

F. Sang, Y. Yang, L. Yuan, J. Ren and Z. Zhang, Nanoscale, 2015, 7, 15852
DOI: 10.1039/C5NR03596A

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