Rapid DNA sequencing by horizontal ultrathin gel electrophoresis

Abstract

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.

DNA sequencing: modular primers assembled from a library of hexamers or pentamers.

“…Closing (8)(9)(10) and reusable capillary gels (11,12) reduce the sequencing electrophoresis time to <1 hr and the detection limit to a few thousand fluorescent molecules per band (10-20 mol), the modular primers can be expected to decrease the time per walking cycle to 1 or 2 hr from the time of several days it now takes.…”

High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer

“…This concern arose because MicroCel gels are relatively thin (50 mm), are cast by a rapid photopolymerization process, and are cast between ªunpassivatedº (uncoated) glass plates which could contribute strong interfacial effects. While an earlier study of comparably thin slab gels revealed no electrophoretic anomalies [76], we decided to carefully re-examine the issue with MicroCel gels. In a later publication, we will present a detailed study of the factors which limit the electrophoretic resolution on MicroCel gels.…”

Human dopamine D4 receptor allele genotyping by ultrathin agarose gel electrophoresis with To-Pro-3 complexation

“…Chen and co-workers [8] reported the use of 0.1±1 mM To-Pro-3 complexing agent in capillary gel electrophoresis-mediated DNA fragment separation using a 640 nm solid-state red diode laser. Combination of the benefits of slab and capillary gel electrophoresis resulted in high-performance ultrathin gel electrophoresis, a novel and highly efficient multilane separation platform which was first introduced for DNA sequencing [9,10] and later for DNA fragment analysis [11]. This technique also enables separation enhancement and labeling of the migrating dsDNA molecules by noncovalent complexation with intercalator dyes, such as ethidium bromide, SYBR Green, Cascade Blue and To-Pro-3.…”