36] Gas chromatography-mass spectrometry analysis of DNA: Optimization of protocols for isolation and analysis of DNA from human blood

Abstract

This chapter shows that 5 ml of whole blood, or packed cells from this volume, is required to extract 100μg DNA, which allows easy analysis of base damage by GC/MS, and storing blood at -20 ° for up to 1 year does not affect DNA damage. Although the DNA extraction protocol followed in this study requires removal of RNA to prevent artifacts in DNA base damage determination, showing a lack of effect with all the other conditions tested, it is not necessary that every DNA extraction procedure follows this pattern. The optimal protocol may depend on sources of reagents and the general level of contamination with prooxidants (e.g., transition metal ions in the water and reagents) in the laboratory concerned. It has subsequently applied this protocol to the investigation of factors affecting oxidative DNA damage in the human body. It is found that steady-state levels of oxidative DNA damage varied widely among healthy individuals. This may be related to a number of endogenous and exogenous factors, including genetic predisposition, exposure to environmental toxins, and antioxidant intake. Levels of DNA damage and repair is also influenced by endogenous rates of free radical production and level of antioxidant defenses, as well as the activity of repair enzymes and the ability to respond positively to increased levels of oxidative stress, including the absorption, processing, and transport of dietary antioxidants to the site of oxidative stress. Collectively, such factors contribute to the overall levels of oxidative stress in the human body and thus DNA damage and, in the long term, perhaps susceptibility to cancer.