Solid-phase reversible immobilization in microfluidic chips for the purification of dye-labeled DNA sequencing fragments.
In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm(-2). The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished off-chip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method.
Xu
Yichuan
Y
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, USA.
Vaidya
Bikas
B
Patel
Ami B
AB
Ford
Sean M
SM
McCarley
Robin L
RL
Soper
Steven A
SA
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes; D005782:Gels; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D005782:Gels; D009838:Oligodeoxyribonucleotides; D004247:DNA