Immunodetection of DNA with biotinylated RNA probes: A study of reactivity of a monoclonal antibody to DNA-RNA hybrids

Francois Coutlée, Linda Bobo, Kumudini Mayur, Robert H Yolken, Raphael P Viscidi

Research output: Contribution to journalArticle

Abstract

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a β-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 106 pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.

Original languageEnglish (US)
Pages (from-to)96-105
Number of pages10
JournalAnalytical Biochemistry
Volume181
Issue number1
DOIs
StatePublished - Aug 15 1989

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RNA Probes
Monoclonal Antibodies
RNA
DNA
Polymerase chain reaction
Viruses
HIV-1
Polymerase Chain Reaction
Nucleic Acids
Assays
HIV
Galactosidases
Taq Polymerase
pol Genes
Fluorescent Dyes
Dilution
Amplification
Plasmids
Genes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Immunodetection of DNA with biotinylated RNA probes : A study of reactivity of a monoclonal antibody to DNA-RNA hybrids. / Coutlée, Francois; Bobo, Linda; Mayur, Kumudini; Yolken, Robert H; Viscidi, Raphael P.

In: Analytical Biochemistry, Vol. 181, No. 1, 15.08.1989, p. 96-105.

Research output: Contribution to journalArticle

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