Solid-Phase Reversible Immobilization in Microfluidic Chips for the Purification of Dye-Labeled DNA Sequencing Fragments

Abstract

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.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips

“…This was the approach used by the group of Regnier who constructed a number of small offset pillars for chromatography and electrochromatography [251][252][253][254][255]. The only report till date on this kind of column for preconcentration is by Xu et al [256] who used modified polycarbonate microchips for the extraction of DNA. While this is an elegant and simple approach, it may be difficult to reproducibly obtain the high aspect ratio and minimal distance between each pillar required for efficient extraction.…”

Purification of Nucleic Acids in Microfluidic Devices

“…37 Micropost-anchored carboxylate groups generated by UV radiation provided an adequate DNA binding surface, although with a relatively low binding capacity relative to other pillar structure devices. Witek et al exploited a similar microfluidic PPC device for genomic DNA purification from whole E. coli cell lysate before PCR.…”

Permanent surface modification of polymeric capillary electrophoresis microchips for protein and peptide analysis

“…Carboxyl moieties can also be produced by ablating the polymer in an oxygen atmosphere with a 248-nm KrF excimer laser [96]. A PC surface can be activated by using UV/O 3 [97] or 254-nm UV radiation [98] to produce hydroxyl and carboxyl groups, respectively. To decrease the hydrophobicity of PCOC, Gaudioso and Craighead [99] used an oxygen plasma to treat the surface.…”