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Digestion of Methylated DNA:

Effect of Dam and Dcm Methylation on DNA Cleavage by Restriction Enzymes

To cleave with a restriction enzyme which is sensitive to the Dam methylation, DNA should be purified from dam- E.coli strains. To cleave with a restriction enzyme which is sensitive to Dcm methylation, DNA should be purified from dcm- E.coli strains. E.coli GM2163 dam-, dcm- (#M0099) is available upon request. Control digestions should be performed with Lambda DNA (dam-, dcm-).

Dam Methylation

Dcm Methylation

Dam Methylation

Enzymes with Recognition Sequences Completely Overlapping Dam Methylase Site GATC

Single letter code:
R = G or A; 
Y = C or T; 
W = A or T; 
M = A or C; 		 K = G or T; 
S = C or G;
H = A, C or T;
V = A, C or G;		 B = C, G or T;
D = A, G or T;
N = G, A, T or C.

A - N6-methyladenine (m6A)
Recognition sequence is indicated in bold.
Overlapping methylase sequence is green shaded.

Enzyme Sequence*

Effect
BamHI
FastDigest® BamHI		 GGATCC not blocked
BclI
FastDigest® BclI		 TGATCA blocked
BglII
FastDigest® BglII		 AGATCT not blocked
Bsp143I GATC not blocked
BspPI GGATC blocked
MboI
FastDigest® MboI		 GATC blocked
PsuI
FastDigest® PsuI		 RGATCY not blocked
PvuI
FastDigest® PvuI		 CGATCG not blocked

Note
DpnI (Gm6^ATC) - cleaves only Dam methylated DNA.

Enzymes with Recognition Sequences which may Partially Overlap Dam Methylase Site GATC

Single letter code:
R = G or A; 
Y = C or T; 
W = A or T; 
M = A or C; 		 K = G or T; 
S = C or G;
H = A, C or T;
V = A, C or G;		 B = C, G or T;
D = A, G or T;
N = G, A, T or C.

A - N6-methyladenine (m6A)
Recognition sequence is indicated in bold.
Overlapping methylase sequence is green shaded.

Enzyme Sequence* Effect*
AloI 5'...GAAC(N)4GATCC...3'
3'...CTTG(N)4CTAGG...5' 		not determined
BdaI 5'...TGATC(N)4TCA...3'
3'...ACTAG(N)4AGT...5' 		blocked
BseJI
FastDigest® BsaBI (BseJI)		 5'...GATC(N)3ATC...3'
3'...CTAG(N)3TAG...5' 		blocked
Bsh1285I 5'...CGATCG...3'
3'...GCTAGC...5'		 not blocked
Bsp68I 5'...GATCGCGATC...3'
3'...CTAGCGCTAG...5'		 not blocked
Bsu15I
FastDigest® ClaI (Bsu15I)		 5'...ATCGATC...3'
3'...TAGCTAG...5'		 blocked
FspBI
FastDigest® BfaI (FspBI)		 5'...CTAGATC...3'
3'...GATCTAG...5'		 impaired
Hin4I 5'...GATC(N)4VTC...3'
3'...CTAG(N)4BAG...5'

5'...GAY(N)4GATC...3'
3'...CTR(N)4CTAG...5'

 blocked


blocked
HphI 5'...GGTGATC...3'
3'...CCACTAG...5'		 blocked
Kpn2I
FastDigest® Kpn2I		 5'...TCCGGATC...3'
3'...AGGCCTAG...5'

5'...GATCCGGATC...3'
3'...CTAGGCCTAG...5'

 not blocked


not determined
MboII
FastDigest® MboII		 5'...GAAGATC...3'
3'...CTT CTAG...5'		 blocked
PagI
FastDigest® BspHI (PagI)		 5'...TCATGATC...3'
3'...AGTACTAG...5'

5'...GATCATGATC...3'
3'...CTAGTACTAG...5'

 impaired


not determined
PfoI
FastDigest® PfoI		 5'...TCCNGGATC...3'
3'...AGGNCCTAG...5'		 impaired
SmoI 5'...CTYRAGATC...3'
3'...GARYTCTAG...5'		 impaired
TaqI
FastDigest® TaqI		 5'...TCGATC...3'
3'...AGCTAG...5'		 blocked
TstI 5'...CAC(N)4GATCC...3'
3'...GTG(N)4CTAGG...5'		 blocked
XbaI
FastDigest® XbaI		 5'...TCTAGATC...3'
3'...AGATCTAG...5'		 blocked

Dcm Methylation

Enzymes with Recognition Sequences Completely Overlapping Dcm Methylase Site CCWGG

Single letter code:
R = G or A; 
Y = C or T; 
W = A or T; 
M = A or C; 		 K = G or T; 
S = C or G;
H = A, C or T;
V = A, C or G;		 B = C, G or T;
D = A, G or T;
N = G, A, T or C.

C - 5-methylcytosine (m5C)
Recognition sequence is indicated in bold.
Overlapping methylase sequence is green shaded.

Enzyme Sequence*

Effect
EcoRII CCWGG blocked
MvaI
FastDigest® MvaI		 CCWGG not blocked
PasI CCCWGGG not blocked
FastDigest® SexAI (CsiI) ACCWGGT not blocked

Enzymes with Recognition Sequences which may Partially Overlap Dcm Methylase Site CCWGG

A    B    C D    E    G H K    M N P R S T V X

Enzyme

Sequence

 Effect
Acc65I
FastDigest® Acc65I		 5'...GGTACCWGG...3'
3'...CCATGGWCC...5'

5'...CCWGGTACCWGG...3'
3'...GGWCCATGGWCC...5'

 impaired

blocked
AloI 5'...GAAC(N)6TCCWGG...3'
3'...CTTG(N)6AGGWCC...5'		 not blocked
ApaI
FastDigest® ApaI		 5'...GGGCCCWGG...3'
3'...CCCGGGWCC...5'		 impaired
BamHI
FastDigest® BamHI

5'...CCWGGATCCWGG...3'
3'...GGWCCTAGGWCC...5'

 not blocked
BfuI 5'...GTATCCWGG...3'
3'...CATAGGWCC...5'		 not blocked
BglI
FastDigest® BglI		 5'...GCCWGGNNGGC...3'
3'...CGGWCCNNCCG...5'		 not blocked
Bme1390I
FastDigest® ScrFI (Bme1390I)		 5'...CCWGG...3'
3'...GGWCC...5'		 blocked
BseDI
FastDigest® BsaJI (BseDI)		 5'...CCWGGG...3'
3'...GGWCCC...5'		 not blocked
BseGI
FastDigest® BseGI		 5'...CCWGGATG...3'
3'...GGWCCTAC...5'		 not blocked
BseLI
FastDigest® BslI (BseLI)		 5'...CCWGGNNNNGG...3'
3'...GGWCCNNNNCC...5'

5'...CCWGGNCCWGG...3'
3'...GGWCCNGGWCC...5'

 impaired

blocked
BseSI
FastDigest® Bme1580I (BseSI)		 5'...GKGCCCWGG...3'
3'...CMCGGGWCC...5'		 not blocked
BshNI
FastDigest® BanI (BshNI)		 5'...GGYRCCWGG...3'
3'...CCRYGGWCC...5'

5'...CCWGGYRCCWGG...3'
3'...GGWCCRYGGWCC...5'

 impaired

not determined
Bsp120I
FastDigest® Bsp120I		 5'...GGGCCCWGG...3'
3'...CCCGGGWCC...5'		 blocked
BspLI
FastDigest® NlaIV (BspLI)		 5'...GGNNCCWGG...3'
3'...CCNNGGWCC...5'

5'...CCWGGNNCCWGG...3'
3'...GGWCCNNGGWCC...5'

 not blocked

blocked
BspPI 5'...CCWGGATC...3'
3'...GGWCCTAG...3'		 not blocked
BstXI
FastDigest® BstXI		 5'...CCAGGNNNNTGG...3'
3'...GGTCCNNNNACC...5'

5'...CCAGGNNCCTGG...3'
3'...GGTCCNNGGACC...5'

 not blocked

blocked
BsuRI
FastDigest® HaeIII (BsuRI)		 5'...GGCCWGG...3'
3'...CCGGWCC...5'

5'...CCWGGCCWGG...3'
3'...GGWCCGGWCC...5'

 not blocked

not blocked
CaiI
FastDigest® AlwNI (CaiI)		 5'...CAGNNCCTGG...3'
3'...GTCNNGGTCC...5'		 blocked
CfrI 5'...YGGCCWGG...3'
3'...RCCGGWCC...5'		 blocked
Cfr13I
FastDigest® Sau96I (Cfr13I)		 5'...GGNCCWGG...3'
3'...CCNGGWCC...5'		 blocked
Eco24I 5'...CCWGGGCCCWGG...3'
3'...GGWCCCGGGWCC...5'		 not blocked
Eco31I
FastDigest® Eco31I		 5'...CCWGGTCTC...3'
3'...GGWCCAGAG...5'		 impaired
Eco47I
FastDigest® AvaII (Eco47I)		 5'...GGWCCWGG...3'
3'...CCWGGWCC...5'		 blocked
Eco91I
FastDigest® Eco91I		 5'...CCWGGTNACCWGG...3'
3'...GGWCCANTGGWCC...5'		 not blocked
Eco57MI 5'...CCTGGAG...3'
3'...GGACCTC...5'		 blocked
Eco147I
FastDigest® StuI (Eco147I)		 5'...AGGCCTGG...3'
3'...TCCGGACC...5'		 blocked
EcoO109I 5'...RGGNCCTGG...3'
3'...YCCNGGACC...5'		 blocked
EheI
FastDigest® EheI		 5'...CCWGGCGCCWGG...3'
3'...GGWCCGCGGWCC...5'		 not blocked
FaqI 5'...CCWGGGAC...3'
3'...GGWCCCTG...5'		 not blocked
GsuI 5'...CTCCAGG...3'
3'...GAGGTCC...5'		 blocked
Hin1I
FastDigest® BsaHI (Hin1I)		 5'...GRCGCCWGG...3'
3'...CYGCGGWCC...5'		 impaired
HphI 5'...CCWGGTGA...3'
3'...GGWCCACT...5'		 not blocked
KpnI
FastDigest® KpnI		 5'...GGTACCWGG...3'
3'...CCATGGWCC...5'

5'...CCWGGTACCWGG...3'
3'...GGWCCATGGWCC...5'

 not blocked

not blocked
MlsI
FastDigest® MscI (MlsI)		 5'...TGGCCAGG...3'
3'...ACCGGTCC...5'		 blocked
PfoI
FastDigest® PfoI		 5'...TCCAGGA...3'
3'...AGGTCCT...5'		 blocked
Psp5II
FastDigest® PpuMI (Psp5II)		 5'...RGGWCCTGG...3'
3'...YCCWGGACC...5'		 blocked
SduI
FastDigest® Bsp1286I (SduI)		 5'...CCWGGGCCCWGG...3'
3'...GGWCCCGGGWCC...5'		 not blocked
SfiI 5'...GGCCWGGNNGGCC...3'
3'...CCGGWCCNNCCGG...5'

5'...CCWGGCC(N)5GGCCWGG...3'
3'...GGWCCGG(N)5CCGGWCC...5'

 impaired

not blocked
Van91I
FastDigest® PflMI (Van91I)		 5'...CCAGGNNNTGG...3'
3'...GGTCCNNNACC...5'		 blocked
TstI 5'...CAC(N)6TCCWGG...3'
3'...GTG(N)6AGGWCC...5'		 not determined
XagI
FastDigest® EcoNI (XagI)		 5'...CCTGGNNNAGG...3'
3'...GGACCNNNTCC...5'

5'...CCTGGNCCAGG...3'
3'...GGACCNGGTCC...5'

 not blocked

not blocked

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Updated rugsėjo 03, 2008 15:07