Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels.
Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.
Xu
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Department of Chemistry, Louisiana State University, Baton Rouge 70803-1804, USA.
Bruch
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RC
Soper
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D010582:Bacteriophage lambda / Q000235:genetics; D019149:Bioreactors; D001710:Biotin; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D005782:Gels; D008832:Microchemistry / Q000379:methods; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D019265:Spectroscopy, Near-Infrared; D019809:Streptavidin
D017931:DNA Primers; D004279:DNA, Viral; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D005782:Gels; D001710:Biotin; D019809:Streptavidin