Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining.

F. Dolbeare, Joe Gray

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.

Original languageEnglish (US)
Pages (from-to)631-635
Number of pages5
JournalCytometry
Volume9
Issue number6
StatePublished - Nov 1988
Externally publishedYes

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Pyrimidines
Single-Stranded DNA
DNA Restriction Enzymes
Staining and Labeling
Deoxyribonuclease EcoRI
Idoxuridine
Endonucleases
Bromodeoxyuridine
Anti-Idiotypic Antibodies
Octoxynol
Nuclear Proteins
Cricetulus
Citric Acid
exodeoxyribonuclease III
Digestion
Ovary
Antibodies
DNA
5-hexyl-2'-deoxyuridine

ASJC Scopus subject areas

  • Biophysics
  • Medicine(all)

Cite this

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining. / Dolbeare, F.; Gray, Joe.

In: Cytometry, Vol. 9, No. 6, 11.1988, p. 631-635.

Research output: Contribution to journalArticle

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