Detection of modified DNA nucleotides by postlabeling procedures

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

During the last 15 years the number of studies using postlabeling techniques to detect molecular alterations in DNA exposed to genotoxic agents has been continuously growing. Detectable molecules no longer include only bulky adducts arising from environmental exposures to polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, cigarette smoke, etc. Postlabeling procedures are also able to reveal small DNA adducts and small nucleotide alterations induced by genotoxic agents such as alkylating compounds and aflatoxins. DNA alterations induced by oxidizing molecules both of exogenous and endogenous source can also be revealed. Postlabeling methods have been successfully used to detect DNA damage induced by ionizing and exciting radiations. Variants of the basic procedure allow the detection of endogenous DNA modifications associated with aging, called I-compounds. Postlabeling methods are able to detect such a great variety of molecular alterations by using a negligible amount of DNA (1-5 μg) with an extraordinary high sensitivity (up to 1/1010 modified nucleoside/normal nucleotides). Different modifications of the basic procedure are applied depending on the specific nucleotidic modification under analysis. The present article describes the main methodological variants of postlabeling techniques, with particular attention paid to their methodological aspects, applications, and capabilities. Each step of the postlabeling procedure (i.e., DNA depolymerization, adduct enrichment, labeling, and identification) is described and the most useful variants currently available are reported. DNA depolymerization may be performed by using at least 5 different nucleases to obtain biphosphate mononucleotide, monophosphate mononucleotide, dinucleotides, or trinucleotides. Modified nucleotides can be selected from among normal nucleotides by enrichment procedures including more than 6 different chemical or enzymatic methods. Adducts are labeled by using radioactive carriers such as AT32 P and other radioisotopes (33P, 35S) or fluorochromes (dansyl chloride). Labeled adducts are separated by thin- layer or column chromatography using a great variety of chromatographic media. Finally, modified nucleotides are revealed and quantified by various techniques, including standard, laser scan, and electronic autoradiography. Thus, the postlabeling procedure no longer can be considered a single toxicologic method. It is a class of analytical tools able to detect a wide variety of nucleotidic modifications induced by genotoxic agents.

Original languageEnglish
Pages (from-to)175-205
Number of pages31
JournalToxicology Methods
Volume8
Issue number3
DOIs
Publication statusPublished - Jul 1998

Fingerprint

Nucleotides
DNA
Depolymerization
DNA Adducts
Thin layer chromatography
Column chromatography
Molecules
Aflatoxins
Polycyclic Aromatic Hydrocarbons
Fluorescent Dyes
Nucleosides
Smoke
Tobacco Products
Radioisotopes
Labeling
Amines
Aging of materials
Environmental Exposure
Radiation
Ionizing Radiation

Keywords

  • P postlabeling
  • DNA adducts
  • Genotoxic damage

ASJC Scopus subject areas

  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Detection of modified DNA nucleotides by postlabeling procedures. / Izzotti, Alberto.

In: Toxicology Methods, Vol. 8, No. 3, 07.1998, p. 175-205.

Research output: Contribution to journalArticle

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