Stepwise partially overlapping primer-based PCR for genome walking

AMB Express

Table 1 Primers used in this study

Primer set	Stepwise partially overlapping primer set (SWPOP primer set)	Specific primer set (SP primer set)^a
Primer set		gadA locus	hyg
Primary PCR	CAGTCAGTCTCAGCTAGTCAGTGTC	TCCATACCCTCATCTCCATTTCCAT (− 1 to − 25)	CGGCAATTTCGATGATGCAGCTTGG (− 1 to − 25)
	CAGTCAGTCTCGTCAGTCGTGCAGT
	CAGTCAGTCTCCACGTCACCAGTCA
	CAGTCAGTCTAGCAGCAGTCGTCAG
Secondary PCR		AACTATCACCCCACAACGTCATCTC (− 157 to − 181)	CGGGACTGTCGGGCGTACACAAATC (− 64 to − 88)
Tertiary PCR		ACCGTTCATAGGCGAAATTGTTTGT (− 335 to − 359)	GACCGATGGCTGTGTAGAAGTACTC (− 112 to − 136)

Four SWPOP-P primers were respectively paired with a specific primer for primary PCR; in the subsequent secondary or tertiary PCR, the SWPOP primer and a corresponding specific primer in the same row were matched. The partial identical sequences of SWPOP primers were underlined
^aThe bracketed numerical ranges below specific primers refer to coordinates in the known DNA sequences (a first base at the 5′-end of an outermost specific primer is defined as-1)