HIGH-SPEED SCREENING OF POLYMERASE CHAIN-REACTION PRODUCTS BY CAPILLARY ELECTROPHORESIS

In an effort to develop capillary electrophoresis (CE) for high-throug hput polymerase chain reaction (PCR) molecular diagnostics, a method w as developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR pro ducts (375 and 400 bp) in an assay of TGF-beta(1) knockout mice to det ermine the genotype of neonates. Using a commercially available CE ins trument, the two PCR products were separated in 12 min with a replacea ble gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm held strength. With the coinjection of a 20-bp ladder, the sizes of t he PCR products were determined hom the electropherogram without using a calibration plot and curve-fitting program, Faster separation was o btained using the combination of a short effective length capillary an d high field strength. The two PCR products were separated in 82 s wit h a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separa tion, with minimum postrun data processing, is highly desirable for th e high-throughput screening of PCR products using a single-capillary C E system. (C) 1996 Academic Press, Inc.