Single molecule spectroscopic investigations of high density labeled DNA with regard to single-molecule DNA sequencing-by-degradation
In the post-genome era, fast and inexpensive ways to obtain sequence-specific information for e. g., comparative genomics, medical diagnostics and several biological applications are required . During the past years, a series of interesting new attempts were made to overcome the limitations of the conventional used Sanger sequencing. One of these ideas is based on the successive enzymatic degradation of a completely labeled target DNA-molecule [2-6]. The fluorescent nucleoside monophosphates are released as a consequence of exonucleolytic hydrolysis, and they can be identified within milliseconds by recording their fluorescence characteristics (wavelengths, fluorescence lifetimes) during passage of a laser focus . This concept seems to be relatively simple, but there are many difficult problems to solve. Fluorescently labeled nucleotides are bulkier and more lipophilic than the natural ones, and thus, confer new chemical properties to the product DNA such as lowered solubility in aqueous media . Further difficult tasks concern the selection of single target DNA molecules and the correct sequen- tial identification of released nucleotides. As a consequence, sequencing of single DNA fragments has not been proven so far. A question that arised is, can enzymatic manipulation of microarrayed DNA combined with single-molecule detection to a system that allows for the real-time observation of DNA degradation and thus, tackle the problems of diminished solubility, spatial separation and allocation of target DNA?