Fast DNA fragment sizing in a short capillary column.

Abstract

Capillary electrophoresis (CE) is an efficient and fast microseparation technique that is well suited for the separation of nucleic acids. CE is routinely used for the separation of double-stranded DNA fragments; single-stranded DNA fragments and polymerase chain reaction (PCR) products in our laboratory. CE is a versatile tool for separating nucleic acids in molecular biology (1). CE has the advantages of automation, small sample requirement, fast and efficient separation, real-time detection, and negligible buffer waste in comparison to slab-gel electrophoresis. However, the major drawback of CE is low amount of sample throughput because samples are analyzed sequentially. This problem is resolved by using a capillary array electrophoresis (CAE) (2,3). Since conventional CE systems are equipped with a single capillary, a practical way to increase throughput is to minimize the analysis time per sample. This has been accomplished by using a range of an effective length capillary as short as 7 cm and field strength as high as 2000 V/cm for the separations of small ions, drugs, and proteins (4–7). Recently, we were able to achieve fast separation of the wild-type and mutant PCR products of the TGF-β1 gene in 60 s using a 7-cm effective length capillary and 560 V/cm (8). However, high field strength degraded the resolution of large DNA fragments (9) and may cause migration anomalies (10). Alternatively, one can use a shorter capillary length with low field strength. We describe here the fast sizing of DNA fragments using 1-, 2-, and 7-cm effective length capillaries (11). Fast DNA separation can be achieved with capillaries with an effective length of only 1–2 cm. The low electric field associated with such short capillaries extends the range of DNA fragment sizes that may be separated by this means, which is useful if the analysis of large DNA fragments is required.