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 assay s. 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 codi ng sequence and partial promoter region of the lacI gene were amplifie d by the polymerase chain reaction with [alpha-P-32]dCTP in a 1247 bas e pair fragment, digested into eight restriction fragments, and analyz ed by SSCP. The sensitivity of the method was assessed using 160 phage s with lacI mutations, which were selected by assay of expression of b eta-galactosidase after their infection into E. coli. Of the 160 mutan ts, 146 (91.3%) showed shifted bands in the first condition of SSCP an alysis (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 muta nts). The remaining six mutants were analyzed in a third condition (wi th 5% glycerol, 10 degrees C), and all of them showed shifted bands (c umulatively 100%). Sequencing of the restriction fragments with mobili ty 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 cond ition. This method greatly reduced the time to identify lacI mutations , and allowed the detection of multiple mutations in one lad mutant, T he results also show that in general PCR-SSCP analysis is very sensiti ve when test fragments are shorter than about 250 base pairs and elect rophoresis is performed under at least two conditions.