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 1 Primers Used for Mutagenesis

HP-NAP mutants	Primer sequence (5′-3′)^{a, b, c}	Tm_pp (°C)^{d, e}	Tm_no (°C)^{d, f, g}	Inserted silent restriction sites	Size of the digested products (bp)^h
HP-NAP_D98A	F: AAATT CTcGAG GctTACAAA TATCTAGAAAAAGAATTTAAAGAGC	54	62	XhoI	5261, 307
	R: TTTGTAagC CTCgAG AATTT CTTTAAAGATGTCTTTAGAGTGG	54	62
HP-NAP_Y99A	F: ATT CTcGAG GACgcCAAA TATCTAGAAAAAGAATTTAAAGAGC	54	62	XhoI	5261, 307
	R: TTTGgcGTC CTCgAG AAT TTCTTTAAAGATGTCTTTAGAGTGG	54	66
HP-NAP_Y101H	F: ACAAAcAT CTcGAg AAAGAA TTTAAAGAGCTCTCTAACACC	54	58	XhoI	5261, 307
	R: TTCTTT cTCgAG ATgTTTGT AGTCCTCTAGAATTTCTTTAAAGA	54	62
HP-NAP_E103A	F: TCTAGcAAAA GAATTc AAAGA GCTCTCTAACACCGCTGAAAA	54	62	EcoRI	5568
	R: TCTTT gAATTC TTTTgCTAGA TATTTGTAGTCCTCTAGAATTTCT	54	62
HP-NAP_E103D	F: ACAAATATCTAGAcAAA GAATT c AAAGAGCTCTCTAACACCGCT	54	62	EcoRI	5568
	R: AATTC TTTgTCTAGATATTTGT AGTCCTCTAGAATTTCTTTAAAGA	54	62
HP-NAP_E97GY101H	F: ACAAAcAT CTcGAg AAAGAA TTTAAAGAGCTCTCTAACACCG	54	62	XhoI	5261, 307
	R: TTCTTT cTCgAG ATgTTTGT AGTCCcCTAGAATTTCTTTAAAGAT	54	66

^aPrimer-primer overlapping sequences are written in bold
^bInserted silent restriction sites are underlined
^cMutations are written in lowercase letters
^dTm_pp and Tm_no were calculated as: Tm = 2 °C x (number of the A and T bases) + 4 °C x (number of the G and C bases)
^eTm_pp was calculated from the primer-primer overlap sequence
^fTm_no was calculated from the primer sequence matched to the template
^gThe Tm_no value of 62 °C was used for the PCR reaction to generate the mutations if the Tm_no values of forward and reverse primers are different
^hThe digested products are DNA fragments from the plasmid with desired mutations after digestion with the inserted silent restriction enzyme

ISSN: 1480-9222