Table 3.

Design of oligonucleotides containing predicted recognition sequences as well as restriction sites for methylation sensitivity assays

R-M system plasmids constructed	Recognition sequence (degeneracy)	Designed oligos	Reasoning behind the methylation sensitivity assay	Predicted digestion of plasmid with type I sequence by the constructed corresponding type II enzyme
				Before modification	After modification
Eco394I	5′GAC(5N)RTAAY3′
	3′CTG(5N)CATTR5′
pEco394AC	(R-Y = A-C)	5′GACGCGTCATAAC3′	MluI does not cut ACGCGT	Cut	Not cut
pEco394AT	(R-Y = A-T)	5′GACTAGTCATAAT3′	SpeI does not cut ACTAGT	Cut	Not cut
pEco394GCr	(R-Y = G-C)	3′CTGATGCGCATTG5′	MluI does not cut ACGCGT	Cut	Not cut
pEco394GTr	(R-Y = G-T)	3′CTGCTGATCATTA5′	SpeI does not cut ACTAGT	Cut	Not cut
Eco826I	5′GCA(6N)CTGA3′
	3′CGT(6N)GACT5′
pEco826		5′GCAGTACTCCTGA3′	ScaI does not cut AGTACT	Cut	Not cut (Fig.3B)
pEco826r		3′CGTCGTCATGACT5′	ScaI does not cut AGTACT	Cut	Not cut
Eco851I	5′GTCA(6N)TGAY3′
	3′CAGT(6N)ACTR5′
pEco851T	(Y = T)	5′GTCAGTACTCTGAT3′	ScaI does not cut AGTACT	Cut	Not cut (Fig.3B)
pEco851Tr	(Y = T)	3′CAGTGTCATGACTA5′	ScaI does not cut AGTACT	Cut	Not cut
pEco851C	(Y = C)	5′GTCAGTACTCTGAC3′	ScaI does not cut AGTACT	Cut	Not cut
pEco851Cr	(Y = C)	3′CAGTGTCATGACTG5′	ScaI does not cut AGTACT	Cut	Not cut
Eco912I	5′CAC(5N)TGGC
	3′GTG(5N)ACCG
pEco912		5′CACTAGTCTGGC3′	SpeI does not cut ACTAGT	Cut	Not cut (Fig.3B)
pEco912r		3′GTGTCATGACCG5′	ScaI does not cut AGTACT	Cut	Not cut

The type I recognition sequences are underlined in the designed oligo column. The type II recognition sites in the same column are shown in bold. Target adenines in each recognition sequence are bold and underlined. Methylated adenines in the ‘Reasoning’ column are underlined. In the cloning experiments, either an SspI site or EcoRV (blunt end) site was added to each designed oligo and ligated into the either unique pMECA SspI site or EcoRV site.