A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2010-03

AUTHORS

Chang Zhang, Yang Wu, Yuna Sun, Chuan Hong, Kehui Xiang, Yu Guo, Mark Bartlam, Zhiyong Lou

ABSTRACT

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions. More... »

PAGES

284-290

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s13238-010-0031-0

DOI

http://dx.doi.org/10.1007/s13238-010-0031-0

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1027318936

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/21203975


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