Assay of deoxy-deazapurines in DNA by 3'-phosphorylation and two-dimensional thin-layer chromatography

J. J. Steinberg, Gary W. Oliver, Antonio Cajigas

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Deoxy-deazapurines (deaza-dNMPs) are incorporated into cellular DNA after administration of anti-neoplastic, anti-viral, or anti-parasitic chemotherapy. Deaza-dNMPs are stable purine analogues and can be detected via 32P-labeling cold DNA. Assay of analogue incorporation and normal base composition is carried out by radiolabeling DNA with all four deoxynucleotides (dNMPs) through nick translation. 3'-Monophosphate digest radiolabels representative dNMPs and deaza-dNMPs. Separation occurs in two-dimensional polyethyleneimine-cellulose thin-layer chromatography, which resolves all dNMPs. The technique was applied to human placental and calf thymus DNA, control and altered calf thymus DNA with cold stoichiometric replacement of deaza-dNMPs to include deoxy-deazaadenosine, deoxy-deazaguanosine, and deoxy-deazainosine. Scintillation detection and densitometry both accurately reflect dNMP content. This technique easily and quickly quantifies the low-molecular-mass deaza-dNMP analogues in DNA. Deaza-dNMP uptake into DNA may reflect clinical chemotherapeutic efficacy and host toxicity. The assay may therefore serve as an early biochemical dosimeter of drug effect and resistance.

Original languageEnglish (US)
Pages (from-to)277-285
Number of pages9
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume612
Issue number2
DOIs
StatePublished - Feb 26 1993

ASJC Scopus subject areas

  • Chemistry(all)

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