Assaying the activity of HIV-1 integrase with DNA-coated plates.

Abstract:

:Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.

journal_name

J Virol Methods

authors

Chang YC,Ching TT,Syu WJ

doi

10.1016/0166-0934(96)02033-2

subject

Has Abstract

pub_date

1996-05-01 00:00:00

pages

135-40

issue

1-2

eissn

0166-0934

issn

1879-0984

pii

0166-0934(96)02033-2

journal_volume

59

pub_type

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