Epi-fluorescence Microscopy of Single Molecule DNA Denaturation in situ

Abstract

AbstractDNA can be denatured by two main methods which are: a) denaturation in solution (invitro) and b) denaturation on a slide surface (in-situ). Additionally, DNA can also be denatured in gels with urea. The method to be used depends on various factors such as the application, the source of the DNA, the length, and the techniques available to confirm the extent of denaturation. Verification of the extent of denaturation is important because of the following factors: 1) increases the chances of hybridization (especially for short probes), 2) prevents the loss of expensive probes (if the target site is not denatured then, the probes will not hybridize and will only cause a high a background), 3) a higher degree of denaturation allows for more probes to be used and therefore, more information can be derived after hybridization, and 4) essential to maximize due to extremely short probe length. It is important to ensure that DNA morphology is preserved after denaturation in order for the probes to hybridise and also for ensuring proper statistical analysis for high throughput applications. In this work, various experimental conditions for in situ denaturation of single molecule DNA is presented.Significance StatementThe significance of this work is that it emphasizes on the importance of denaturation of target genomic DNA in DNA fibre FISH (fluorescence in situ hybridisation) experiments. If the quality of the target DNA is poor after denaturation or the target DNA is not properly denatured, then it will be very difficult or impossible to hybridize the probe DNA during FISH experiments. This will affect the final results for DNA FISH. Additionally, it is the first time that single DNA combed molecules have been shown to be denatured in situ. Most of the past work has been on gels only. Thus the work is both unique and significant.