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Abstract
We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.
Highlights
It is known that tens of thousands of lesions occur daily in each human cell [1]
Several methods are used to assess the mutagenic potential of a specific DNA lesion, with the most concerning lesions produced by oxidative stress
7,8-dihydro-8-oxoguanine (8-oxoG), is important because it is widely present in DNA [4,5]
Summary
It is known that tens of thousands of lesions occur daily in each human cell [1]. Obviously, the development of such lesions into fully realized mutations is one of the key moments in the pathogenesis of numerous diseases [2,3]. The advantage provided by this approach is that the “mirror” products (DNA strands complementary to the starting template) may contain a substitution mutation opposite the lesion, but other sorts of mutations as well, either opposite the lesion or somewhere else in the strand. Each of those mutations will produce different nucleotide sequences, allowing their separation and subsequent identification, making for more informative results
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