Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Table 1

List of primers used for COE-PCR showing restriction sites and base substitutions.

Primer	Sequence ()	Primer length (bp)	(°C)	Length of overlapping bases (bp)
FGSP1	GAAGTCGCAACTAGTTTCTGACATGGAAAAC	31	60	—
RGSP1	CGAAAAACCCCGGGAAAGGGAGG	23	61	—
FGSP2	GCGGTTCCATGGGTCTAAGG	20	58	—
RGSP2	GGATCCGATATCAGCCATGGTTTTT	25	58	—
FMP1	GCTAAGGTGATtcGcCGTGTTATTCC	26	61	26
RMP1	GGAATAACACGgCgaATCACCTTAGC	26	61
FMP2	CCTATGGAGAAACTgATtATTTACCGCTTAAA	32	61.3	32
RMP2	TTTAAGCGGTAAATaATcAGTTTCTCCATAGG	32	61.3
FMP3	TTGCAATTACCCgATtGTCCAACAAATG	28	59.4	28
RMP3	CATTTGTTGGACaATcGGGTAATTGCAA	28	59.4
FMP4	CTTGTTGcGtGGGTTCA	17	45.2	17
RMP4	TGAACCCaCgCAACAAG	17	45.2
FMP5	TCACTGGcGtcGcTGGGCAG	20	46.5	20
RMP5	CTGCCCAgCgaCgCCAGTGA	20	46.5
FMP6	CATTGTCCTgGAGcGtCGcCTTAAACAA	28	41.9	28
RMP6	TTGTTTAAGgCGaCgCTCcAGGACAATG	28	41.9
FMP7	GAGTAATTTAcGcATGcGtcGcAGTGATCTTC	32	<40	32
RMP7	GAAGATCACTgCgaCgCATgCgTAAATTACTC	32	<40
FMP8	GCCCCCTTCTAcATtagtTTtTATATGAAGTC	32	45	32
RMP8	GACTTCATATAaAAactaATgTAGAAGGGGGC	32	45

Underlined are restriction sites. Substituted bases are bold and written in lowercase.