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

David J. Kemp, Melissa J. Churchill, Donald B. Smith, Beverley A. Biggs, Simon J. Foote, M. Gregory Peterson, Nick Samaras, Nicholas J. Deacon, Richard Doherty

Research output: Contribution to journalArticleResearchpeer-review

32 Citations (Scopus)

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 simultaneouly, 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.

Original languageEnglish
Pages (from-to)223-228
Number of pages6
JournalGene
Volume94
Issue number2
DOIs
Publication statusPublished - 1 Jan 1990
Externally publishedYes

Keywords

  • amplified DNA assay
  • avidin-peroxidase
  • biotin
  • diagnostics
  • DNA-binding protein GCN4
  • glutathione S-transferase
  • oligodeoxyribonucleotides
  • Recombinant DNA
  • Taq-polymerase

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