FIG. 2.

Schematic representation of the strategy for overlapping PCR introducing mutations into rlip. M1 and M2 are mutation sites; M1R and M2R represent overlap primers; M1F and M2F represent primers containing mutation sites. In the first-step PCR, two fragments for single mutation or three fragments for double mutation are amplified. After overlap extension (exten.) PCR, the reconstituted mutant genes are further amplified in the second-step PCR by a homoprimer, T2. The sequences of the primers in the experiment are listed here: T2, GCGTACTAGCGTACCACGTG; F2-T2, GCGTACTAGCGTACCACGTGATCTCGATCCCGCGAAATTAATACGACTCAC; R2-T2, GCGTACTAG CGTACCACGTGGCCAGATCCGGATATAGTTCCTCCTTTCAG; C190R, ACTGCCCGGCGCACCCAGGC; C190SF, GCCTGGGTGCGCCGGGCAGTTCGCAGACCGGCGCGCCGACCGA; C270R, CTTCGACACGAGCCCGTCGTTCT; C270SF, AGAACGACGGGCTCGTGTCGAAGTCGAGTGCG CTGTACGGCAAGGTGC; D242R, GAGCACGTTCGCCAGATCAA; D242SF, TTGATCTGGCGAACGTGCTCGCGCCGTCGACGCTCGCGCTGTT; D288R, GAGGTGGTTCCACTTGTAGCTC; D288AF, GAGCTACAAGTGGAACCACCTCGCGGAGATCAACCAGCTGCTCGGCG.