Research Project
6074331
The goal of this project is to develop new media for DNA sequencing by slab gel and capillary electrophoresis which increase the read length at high accuracy obtainable on the commercially available automated DNA sequencers by at least 25 percent. These increases in read length will directly translate into increased throughput of large genome sequencing facilities, both by increasing the data available from each sequencing reaction, and decreasing the number of overlapping sequencing reactions necessary for each "contig". These new electrophoretic media will be based on new, novel vinyl monomers of increased hydrolytic stability and hydrophilicity invented by the applicant organization. Previously, little substantially new gel or polymer chemistry has been brought to bear on high throughput DNA sequencing. New gel formulations based on the novel monomers will be rigorously tested for robust performance (read-length and accuracy) on laboratory-scale commercial sequencing apparatus and in a high-throughput genome sequencing facility. High molecular weight polymers from these monomers will be synthesized by emulsion polymerization, and these will be tested both in laboratory CE instrumentation on production sequencing capillary array sequencers. In addition to read-length, capillary polymers will be scored on their ability to suppress EEO by acting as self-coating sieving media. Read-length and accuracy will be scored using software packages which have wide acceptance in the genome community, such as PHRED and BaseFinder. Compatibility with various dye chemistries will be insured by measuring read lengths for all of the commonly used cycle sequencing labeling schemes. These new electrophoresis media will increase the speed of genome sequencing, and single nucleotide polymorphism mapping by sequencing.
