Journal
ELECTROPHORESIS
Volume 32, Issue 22, Pages 3233-3240Publisher
WILEY-BLACKWELL
DOI: 10.1002/elps.201100260
Keywords
Blended polymer; Chip electrophoresis; Design of experiments; Large DNA separation
Funding
- National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH) [5 R01 HG002918-01]
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Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of similar to 27?kDa and similar to 1?MDa were blended with a second class of polymer, high-molar mass (similar to 7?MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (20040?000?bp) was completed in 2?min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1?kbp, medium dsDNA fragments between 1 and 10?kbp, and large dsDNA fragments above 10?kbp.
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