Table 1.

Primers Used for Mutagenesis

HP-NAP mutants	Primer sequence (5′-3′)a, b, c	Tmpp (°C)d, e	Tmno (°C)d, f, g	Inserted silent restriction sites	Size of the digested products (bp)h
HP-NAPD98A	F: AAATT CTcGAG GctTACAAA TATCTAGAAAAAGAATTTAAAGAGC	54	62	XhoI	5261, 307
	R: TTTGTAagC CTCgAG AATTT CTTTAAAGATGTCTTTAGAGTGG	54	62
HP-NAPY99A	F: ATT CTcGAG GACgcCAAA TATCTAGAAAAAGAATTTAAAGAGC	54	62	XhoI	5261, 307
	R: TTTGgcGTC CTCgAG AAT TTCTTTAAAGATGTCTTTAGAGTGG	54	66
HP-NAPY101H	F: ACAAAcAT CTcGAg AAAGAA TTTAAAGAGCTCTCTAACACC	54	58	XhoI	5261, 307
	R: TTCTTT cTCgAG ATgTTTGT AGTCCTCTAGAATTTCTTTAAAGA	54	62
HP-NAPE103A	F: TCTAGcAAAA GAATTc AAAGA GCTCTCTAACACCGCTGAAAA	54	62	EcoRI	5568
	R: TCTTT gAATTC TTTTgCTAGA TATTTGTAGTCCTCTAGAATTTCT	54	62
HP-NAPE103D	F: ACAAATATCTAGAcAAA GAATT c AAAGAGCTCTCTAACACCGCT	54	62	EcoRI	5568
	R: AATTC TTTgTCTAGATATTTGT AGTCCTCTAGAATTTCTTTAAAGA	54	62
HP-NAPE97GY101H	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