. 2004 Apr 15;32(7):e60. doi: 10.1093/nar/gnh054

Table 2. PCR primers, annealing temperature, product size and restriction enzymes for RFLP.

No.	PCR primer 1 (5′–3′)	PCR primer 2 (5′–3′)	Annealing temp. (°C)	Product size (bp)	Restriction enzyme
A	GTGTAACCCATAACCCCCAAGA	CACCAGCAGACCCTCAAGC	60	226	Eco57I
B	CTGATCTGCTTCTCCCACGAG	GCAGTAAGAGAGTCCGAAATGG	60	243	BtgI
C	TCAACTACGAACTCCCTAATGCAA	GATGACGAGTATGTTTCCAGCAA	60	252	MspI
C(1)^a	TCAACTACGAACTCCCTAATGCAA	GAGGCACCCTTCACAGGAAA	60	147	–
C(2)^a	TCAACTACGAACTCCCTAATGCAA	TTATAGTTTGTTTGCCCCCTGA	60	477	–
D	TGAAATATGTTGCCACTTACCTACTG	CCCAGAGCTGGAAATTGTCATA	60	309	BamHI
E	GGGATGCTTGGAACAGATCAC	CTTGACACAGGGGAGTGGAAG	60	343	BslI
F	GACTGACCCCTATTCCCTGCTT	ACACAGCCAAGGAGATGACAAAG	60	275	DdeI
G	CAGAGCTTGGCATATTGTATC	GTAAACACACAACTAGTCAATG	49	321	SmaI
H	TACCTGCTGTGCACTTGTCC	CCCAATTCTGCACAGTCTGA	60	295	ScaI
I/J	ACCTGATTTCCTTACTGCCTCTTGC	GTCCTGCTTGCTTACCTCGCTTAGT	60	200	BcgI^b/Cfr101
K/L	CAAAAATGAGCACGCTTTCCT	GGCTTTGCTTTGTCTTGTTGC	60	284	DdeI

^aPCR products of experiments C(1) and C(2) were used in the validation of the method.

^bFor RFLP analysis in experiment I, the amplified 200 bp DNA fragment was again amplified with primer 2 and a mutagenesis primer (5′- TACTGGGACGGAACACGATTGAGGTG-3′) to generate a BcgI site in the wild-type fragment.