| Summary: | Carcinogens have shown a sequence specificity in the formation of DNA adducts. Understanding this sequence specificity is an important part of understanding the role which the carcinogen adduct plays in the onset of cancer. The project described in this report addresses the requirement to be able to sequence short oligonucleotides so as to further investigate this sequence specificity. Conventional DNA sequencers to not have the ability to sequence modified DNA strands, and so the use of mass spectrometry is required to enable the location of DNA modifications. Several methods have been investigated in this research. The use of enzymes to generate sequence ladders, followed by HPLC separation and detection of the products by mass spectrometry, allowed full sequence analysis of unmodified and modified oligonucleotides, and the modifications were correctly located for two methylated and one para-benzoquinone-modified DNA bases. The use of tandem mass spectrometry has also been shown to be useful in sequencing DNA strands. In this study, four different instruments have provided data that enables the sequence analysis of differing DNA strands. An electrospray ionisation source was used with three different mass analysers, and in each case, fragmentation patterns observed gave molecular assignments that enabled the full sequence analysis of a range of oligonucleotides up to 10-mer in size, and also of a para-benzoquinone-modified oligonucleotide. Furthermore, the use of the post source decay phenomenon available on a curved field reflectron time of flight instrument allowed full sequence analysis of three 7-mer oligonucleotides by using a matrix assisted laser desorption/ionisation source.
|
|---|
