Application of Shotgun DNA Mapping to Yeast Genomic DNA Shotgun Clones

Shotgun DNA mapping (SDM) is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome. We have previously demonstrated proof of principle for shotgun DNA mapping by using plasmid pBR322 unzipping data amongst yeast genome background[cite preprint]. Currently we are validating the technique using unzipping data from yeast genomic DNA. Genomic DNA from yeast (S. cerevisiae) has been digested with restriction endonucleases to produce a library of random fragments, which we used to create a limited library of shotgun clones. Single-molecule unzipping constructs derived from these clones will be unzipping with optical tweezers (OT). In parallel, we have created a library of simulated possible unzipping force profiles, based on the known yeast genome sequence. The OT data and the library will be used in our existing SDM algorithms to identify each shotgun clone, and success rate will be determined via DNA sequencing of the clones. A major application of SDM we are working towards is mapping of nucleosomes and RNA Polymerase II molecules on native chromatin. We will report our progress towards this goal and also discuss other applications of SDM, including splice variant and telomere analysis.