Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50degreesC, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67degreesC and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality qgreater than or equal to20 (accuracy greater than or equal to99%). This fully integrated nanoliter process decreases the purification time similar to10-fold and the process volume similar to100-fold while providing state-of-the-art sequencing results.