32P post labeling

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The 32 P post labeling is a method for the detection of DNA adducts .

Single step

Enzymatic hydrolysis

The first step involves enzymatic hydrolysis of the DNA using nuclease and phosphodiesterase . The products are 3'-monophosphates. If the DNA contained DNA adducts, a mixture of adducts and adduct-free nucleotides is formed .

Adduct enrichment

Enrichment is optional, but increases sensitivity. In a second step, a selective P1 nuclease cleaves the 3'-phosphate from the normal nucleotides. The nucleotides modified by adducts are resistant to degradation, so that enrichment occurs.

Alternatively, it is also possible to extract the adducts with a hydrophobic structure by means of n-butanol extraction and thus enrich them.

Phosphorylation

To mark the nucleotides, T4 polynucleotide kinase transfers a radioactive phosphate residue from [γ- 32 P] - ATP to the nucleotides in the batch. Deoxyribonucleoside 3 '- [5'- 32 P] bisphosphates are formed.

Separation and evidence

Detection can now take place by means of multi-dimensional thin layer chromatography and scintillation or by means of high-performance liquid chromatography (HPLC). The detection limit is 1 adduct in 10 10 N.

literature

  • Ramesh C. Gupta: Enhanced Sensitivity of 32P-Postlabeling Analysis of Aromatic Carcinogen: DNA Adducts . in: Cancer Research , 45, 5656-5662, November 1985, ( PDF ).
  • MV Reddy and K. Randerath: Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. in: Carcinogenesis 7 (1986) 1543-1551. PMID 3017601
  • DH Phillips: Detection of DNA modifications by the 32P post-labeling assay. in: courage. Res., 378 (1997) 1-12. PMID 9288880
  • K. Randerath and E. Randerath: P-32 postlabeling methods for DNA adduct detection - overview and critical-evaluation. in: Drug Metab. Rev., 26 (1994) 67-85. PMID 8082582
  • K. Randerath, MV Reddy and RC Gupta: 32P-labeling test for DNA damage. in: PNAS , 78 (1981) 6126-6129. PMID 7031643
  • A. Weston: Physical methods for the detection of carcinogen-DNA adducts in humans. in: courage. Res., 288 (1993) 19-29. PMID 7686262
  • A. Weston and MC Poirier in HA Milman and EK Weisburger (Editors): "Handbook of Carcinogen Testing".