Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients.

Important note: Information in this article was accurate in 1991. The state of the art may have changed since the publication date.

Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients.

Gene. 1990 Oct 15;94(2):223-8. Unique Identifier : AIDSLINE MED/91078643
Kemp DJ; Churchill MJ; Smith DB; Biggs BA; Foote SJ; Peterson MG; Samaras N; Deacon NJ; Doherty R; Walter and Eliza Hall Institute of Medical Research, Victoria,; Australia.

Abstract: We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.

Keywords: Acquired Immunodeficiency Syndrome/*GENETICS Cells, Cultured Colorimetry/*METHODS DNA-Binding Proteins/DIAGNOSTIC USE Enzyme-Linked Immunosorbent Assay Fungal Proteins/DIAGNOSTIC USE Glutathione Transferases/DIAGNOSTIC USE Human HIV/*GENETICS In Vitro Polymerase Chain Reaction Support, Non-U.S. Gov't Transcription Factors/DIAGNOSTIC USE JOURNAL ARTICLE
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M9140645

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