A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity.
The lacI gene has been used as a target gene in various mutation assays. We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method. The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with [alpha-32P]dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP. The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E. coli. Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C). The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants). The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%). Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition. This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant. The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions.
Ushijima
T
T
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Hosoya
Y
Y
Suzuki
T
T
Sofuni
T
T
Sugimura
T
T
Nagao
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D056968:Lac Repressors; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins