Restriction mapping of genes by capillary electrophoresis with laser-induced fluorescence detection.
Restriction mapping is one of the essential steps in gene analysis and molecular biology studies. Slab gel electrophoresis is the traditional way to separate DNA fragments for restriction mapping. However, slab gel electrophoresis does not provide sufficient resolution as required in many mapping applications, and the use of radioisotopes in traditional mapping methods creates health hazards. In the present study, capillary electrophoresis coupled with laser-induced fluorescence detection and a modified partial digestion mapping procedure was developed to map DNA fragments. By using capillary electrophoresis, a restriction map of genomic lambda phage clone of human interleukin 5 receptor alpha chain (IL5R alpha) gene was constructed. The IL5R alpha gene was analyzed to have five XbaI enzyme cutting sites at locations 1370, 2290, 2950, 5430, and 9330. The system was further characterized by using pBluescript SK(+) phagemid DNA as a model. Using a sequence-derived map as a reference, the pBluescript SK(+) restriction map constructed by capillary electrophoresis had an accuracy greater than 90%.
Peck
K
K
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
Wung
S L
SL
Chang
G S
GS
Yen
J J
JJ
Hsieh
Y Z
YZ
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007834:Lasers; D018123:Receptors, Interleukin / Q000235:genetics; D053649:Receptors, Interleukin-5; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence
D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D004247:DNA