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

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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 via32P-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. © 1993.

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Steinberg, J. J., Oliver, G. W., & Cajigas, A. (1993). Assay of deoxy-deazapurines in DNA by 3’-phosphorylation and two-dimensional thin-layer chromatography. Journal of Chromatography B: Biomedical Sciences and Applications, 612(2), 277–285. https://doi.org/10.1016/0378-4347(93)80174-3

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