Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Abstract

A new method for rapidly detecting restriction enzyme patterns of Mycobacterium DNA using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439-bp fragment of a 65,000-kDa (M r ) heat shock protein gene (hsp65) of Mycobacterium. After digesting amplification products by BstEII and HaeIII, patterns of enzyme cleavage products were detected by both CE-LIFD and agarose gel electrophoresis (AGE), respectively. Experimental parameters of CE were optimized. Restriction enzyme patterns of Mycobacterium DNA were detected in optimum electrophoresis conditions: a coated capillary column with a length of 50 cm and an internal diameter of 100 μm, an electrophoresis buffer of 45 mmol/l Tris-boric acid-ethylenediaminetetraacetic acid, and a running voltage of 11 kV. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of DNA segments were <3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means to DNA restriction enzyme pattern analysis.