[Restriction enzyme pattern analysis of mycobacteria DNA by capillary electrophoresis with laser induced fluorescence detection].
A new method for rapidly detecting restriction enzyme pattern of mycobacterium deoxyribonucleic acid (DNA) by capillary electrophoresis with laser induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439 bp fragment of 65,000 (Mr) heat shock protein gene (hsp65) of mycobacterium. After digesting the amplification products by BstE II and Hae III respectively, the patterns of enzyme cleavaged products were detected by both CE-LIFD and agarose gel electrophoresis (AGE). The experimental parameters of CE were optimized. The restriction enzyme patterns of mycobacterium DNA can be detected under the optimum electrophoresis conditions: a coated capillary column with the length of 50 cm and 100 microm i. d., electrophoresis buffer of 45 mmol/L TBE (trihydroxymethyl aminomethane (Tris)-boric acid-ethylenediaminetetraacetic acid (EDTA)) and 11 kV running voltage. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of the DNA segments were less than 3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means for DNA restriction enzyme pattern analysis.
Li
Yuanqian
Y
West China School of Public Health, Sichuan University, Chengdu 610041, China. liyuanqian@hotmail.com
Wang
Guoqing
G
Mi
Jianping
J
Zhou
Ying
Y
Zeng
Hongyan
H
Zhang
Chaowu
C
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D018834:Chaperonin 60 / Q000032:analysis; D004262:DNA Restriction Enzymes / Q000032:analysis* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D009161:Mycobacterium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping / Q000379:methods*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases