Development of a High-Throughput Capillary Electrophoresis Protocol for DNA Fragment Analysis

Since the first descriptions of electrophoresis in small diameter tubes in the 1970s and 1980s (
1
,
2
), capillary electrophoresis (CE) has been recognized for its potential to replace slab-gel electrophoresis for the analysisof nucleic acids (
3
,
4
). In particular, the availability of commercial instrumentation for CE over the last several years has made both the sizedetermination and quantitation of
DNA
restriction fragments or polymerase chain reaction (
PCR
) products amenable to automation. Due to the same charge-to-mass ratio, the electrophoretic mobility of nucleic acid molecules in free solution is largely independentof their molecular size (
5
). Therefore, a sieving medium is required for the electrophoretic analysis of DNA fragments based on their size. Typically,two different principal types of separation matrix are used. The first type of matrix is of high viscosity polymer (e.g.,polyacrylamide) with a well-defined crosslinked gel in regard to the structure and size of its pores. The second type of matrixis a noncrosslinked linear polymer network of materials such as, linear polyacrylamide, agarose, cellulose, dextran, poly(ethyleneoxide), with lower viscosity than the former type and with a more dynamic pore structure. Although the first type of matrixis attached covalently to the capillary wall and may provide better separation for small (sequencing) fragments, the secondmatrix format has the advantage of being able to be replenished after each electrophoretic cycle. This typically extends thelifetime of a capillary, prevents contamination of the system, avoids sample carryover and allows the use of temperatureswell above room temperature. Most matrices used in both systems are tolerant to the addition of DNA denaturants. Many differentmedia useful for the separation of DNA have now become commercially available (
6
).