Shotgun DNA Mapping by Unzipping

Reference number: STC-PS-0930
Inventor(s): L. Herskowitz; S. Koch
For more information, contact: Jovan Heusser, M.B.A. (505-272-7908) or Erin M. Beaumont (505-272-7912)

Patent(s)

Application(s) pending

Background

Shotgun DNA Mapping (SDM) is the name given to this newly developed method of unzipping fragments of genomic DNA and identifying said fragments based on their sequence-dependent unzipping forces. A key enabler of SDM is the ability to assign the individual fragments to their specific sites in the genome, based on comparison of the sequence-dependent unzipping force to a library of simulated unzipping forces for all possible fragments. The current innovation is not the data or data acquisition method, but the algorithm, software, and methods developed for identifying particular DNA fragments from an organism whose genome has been sequenced.

Technology

In regards to DNA, unzipping refers to separating the individual strands that comprise the double helix structure of double-stranded DNA. For this application, the optimal method of unzipping is at a constant stretch rate. The force required to unzip the DNA depends on the sequence-dependent energies of the Watson-Crick base pairing (hydrogen bonding) and the base stacking (van der Waals). It has been shown in many independent studies that the unzipping force versus unzipping index is very well predicted by a relatively simple statistical mechanical model. For DNA from a given genome, a site-specific restriction endonuclease will produce a limited number of fragments, each with a “sticky end” that can be ligated onto a common “unzipping construct.” For an organism with a published genome, all possible fragments from the restriction digestion can be identified and their unzipping force pattern can be simulated. Single-molecule unzipping of random fragments produces a force pattern that can be compared to the simulation library. The best match score indicates the identity of the single fragment that was unzipped. Thus, even though direct sequencing by unzipping is not possible, sequence information is effectively obtained based on prior whole genome sequencing work. Variations in the method will allow high-resolution mapping of proteins (e.g. nucleosomes and polymerases) in exact genome locations.

Applications/Advantages

•Mapping protein-DNA interactions on single-chromatin fragments from living cells, for example nucleosomes and polymerases.
•High-throughput structural DNA mapping—single-molecule identification of deletions, insertions, SNPs, and other variation.
•Genome wide mapping of sequence-specific DNA binding proteins—identifying binding sites using digested genomic DNA and purified protein.
•Alternative Splicing—single-molecule splice variant identification via unzipping of whole cell cDNA preps.

Related Categories

• Other Assay Methods and Research Tools