Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Table 2 Analysis of the Mutagenesis Efficiency of the Modified PCR-based Site-directed Mutagenesis Method

Mutated plasmids	Transformed colonies	Silent restriction enzyme digestion			DNA sequencing
Mutated plasmids	Transformed colonies	Plasmids screened^a	Desired plasmids	Mutagenesis efficiency^b	Plasmids screened^a	Desired plasmids	Mutagenesis efficiency^b
HP-NAP_D98A	1	1	1	1/1 (100%)	1	1	1/1 (100%)
HP-NAP_Y99A	3	3	3	3/3 (100%)	1	1	1/1 (100%)
HP-NAP_Y101H	4	4	2	2/4 (50%)	1	1	1/1 (100%)
HP-NAP_E103A	40	3	1	1/3 (33%)	1	1	1/1 (100%)
HP-NAP_E103D	2	2	2	1/2 (50%)	1	1	1/1 (100%)
HP-NAP_E97GY101H	28	4	3	3/4 (75%)	1	1	1/1 (100%)

^aThe plasmids screened by silent restriction enzyme digestion were isolated from randomly selected clones, whereas the plasmids screened by DNA sequencing were randomly selected from the desired plasmids identified by silent restriction enzyme digestion
^bMutagenesis efficiency was calculated as the number of desired plasmids out of a number of plasmids selected for screening

ISSN: 1480-9222