Separation of 4-color DNA sequencing extension products in noncovalently coated capillaries using low viscosity polymer solutions.
A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing in 50 microm capillary at 42 degrees C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA-capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high-throughput instruments for DNA sequencing.
Madabhushi
R S
RS
Perkin Elmer Corporation, Applied Biosystems Division, Foster City, CA, USA. madabhushi1@llnl.gov
D000178:Acrylamides / Q000138:chemical synthesis; D000180:Acrylic Resins / Q000138:chemical synthesis; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D009995:Osmosis; D011108:Polymers / Q000138:chemical synthesis; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions; D013696:Temperature; D014783:Viscosity
D000178:Acrylamides; D000180:Acrylic Resins; D011108:Polymers; D012996:Solutions; D004247:DNA