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 50 degrees C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67 degrees C 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 q > or = 20 (accuracy > or = 99%). This fully integrated nanoliter process decreases the purification time approximately 10-fold and the process volume approximately 100-fold while providing state-of-the-art sequencing results.
Paegel
Brian M
BM
Department of Chemistry, University of California, Berkeley 94720, USA.
Yeung
Stephanie H I
SH
Mathies
Richard A
RA
D000465:Algorithms; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008838:Microcomputers; D036103:Nanotechnology; D009841:Oligonucleotides / Q000032:analysis; D017422:Sequence Analysis, DNA
D009841:Oligonucleotides; D004247:DNA