A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening
Protein & Cell
; (12): 284-290, 2010.
Article
in En
| WPRIM
| ID: wpr-757727
Responsible library:
WPRO
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.
Full text:
1
Index:
WPRIM
Main subject:
Pharmacology
/
Thymine Nucleotides
/
In Vitro Techniques
/
HIV
/
Sequence Analysis, DNA
/
Colorimetry
/
Diphosphates
/
Reverse Transcriptase Inhibitors
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Anti-HIV Agents
/
Nevirapine
Type of study:
Diagnostic_studies
/
Screening_studies
Limits:
Humans
Language:
En
Journal:
Protein & Cell
Year:
2010
Type:
Article