Analysis of Environmentally Induced DNA Damage by Capillary Electrophoresis

Published

September 1, 1999

Author(s)

Z E. Nackerdien, B Siles, S Nivens, Donald H. Atha

Abstract

Chemical pollutants, ionizing radiation, the products of aerobic metabolism and UV light are important metabolic and environmental factors contributing to DNA damage. Ionizing radiation, in paticular, induces damage in the form of single strand breaks, double strand breaks, crosslinks and associated conformational changes to the higher-order structure of DNA inside the cell. Several techniques, such as the single cell gel assay are used to analyze strand breakage in subpopulations of cells. Capillary electrophoresis (CE), by virtue of its high -separation effeciency, throughput, and automation has become recognized as a powerful tool to detect base, sequence and structural changes in nanogram quantities of different DNA substrates. CE can also be combined with mass spectrometry (MS) to detect precursors to DNA strand breaks, I.e., DNA base modifications in human cancer cells (see refs. 1 and 2). In this chapter we describe methods for the analysis of environment-induced structural changes in linear, plasmid and genomic DNA. We describe the response of DNA to graded doses of laser or ionizing radiation as examples of how these methods can be used in a practical manner. Using CE to detect small radiation-induced fragments of DNA and conformational changes in large DNA fragments and plasmids provides a faster and more efficient alternative to other techniques such as standrd and pulsed-field agarose gel electrophoresis. CE can also serve as quality control tools to rapidly monitor the effects of environment-induced DNA damage (2-10). This is particularly useful for large plasmids ({nearly equal to or equal to} 27,000 bp), which are rountinely used in molecular biology and DNA repair assays. Environment-induced DNA damage leads to programmed cell death, otherwise known as apoptosis. CE can be used to monitor the signature pattern of apoptosis, nucleosomal laddering of DNA. The methods described in this chapter are designed to help the development of more efficient strategies for studying mutagenesis or apoptosis. These improved strategies will shed more light on the contributions and levels required of various types of DNA structural damage to apoptosis.

Citation

Methods in Molecular Biology

Volume

162

Pub Type

Journals

Keywords

capillar electrophoresis, DNA damage

Citation

Nackerdien, Z. , Siles, B. , Nivens, S. and Atha, D. (1999), Analysis of Environmentally Induced DNA Damage by Capillary Electrophoresis, Methods in Molecular Biology (Accessed April 18, 2024)