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DNA methylation is the process of transferring a methyl group from a donor molecule to either a cytosine or an adenine by DNA methyltransferases. Such methylation is the most common and abundant DNA modification process in living organisms. Three types of methylated bases are predominantly found in DNA:
Other modified bases, such as 5-hydroxymethylcytosine (hm5C) and 5-hydroxymethyluracil (hm5U), have also been described. The organism-specific pattern of methylation depends on the methyltransferases' specificity.
In prokaryotes, DNA cleavage by a cognate restriction enzyme is prevented by the methylation of DNA by a sequence-specific methyltransferase which is an integral component of every restriction-modification (R-M) system (1, 2).
The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases – Dam and Dcm (3, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (5, 6). The methylase encoded by the dcm gene methylates the C5-position of the internal cytosine residue within the CCWGG sequence (4, 7). To cleave with a restriction enzyme which is sensitive to the Dam or Dcm methylation, DNA should be purified from dam-, dcm- E.coli strains respectively. Control digestions should be performed with Lambda DNA (dam-, dcm-), #SD0021.
In addition to Dam and Dcm methylases, laboratory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8TGCT for EcoBI (3, 4).
DNA from higher eukaryotic organisms possesses modified 5-methylcytosine residues within CpG or CpNpG contexts (2, 8-10). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation.
Most restriction enzymes are sensitive to DNA methylation. In cases where a restriction enzyme target site overlaps a methylation site, the following digestion results are possible:
The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation. For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (11), while the isoschizomer Bsp143I is insensitive to Dam methylation. EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (12). Thus, to achieve effective DNA digestion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction enzyme to that type of methylation.
All restriction enzymes produced by Thermo Scientific have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA.
Enzyme couple | Recognition and cleavage sites | Sensitivity to methylation |
---|---|---|
Acc65I | G^GTACC | Overlapping Dcm or CpG methylation may influence DNA cleavage |
KpnI | GGTAC^C | Not influenced by Dcm or CpG methylation |
ApaI | GGGCC^C | Overlapping Dcm or CpG methylation may influence DNA cleavage |
Bsp120I | G^GGCCC | Blocked by overlapping Dcm or CpG methylation |
Bsp143I | ^GATC | Not influenced by dam, blocked by CpG methylation |
MboI | ^GATC | Blocked by Dam methylated DNA |
DpnI | GA^TC | Cleaves only Dam methylated DNA |
BspOI | GCTAG^C | Not influenced by CpG methylation |
NheI | G^CTAGC | Overlapping CpG methylation may influence DNA cleavage |
Cfr9I | C^CCGGG | CpG methylation may influence DNA cleavage |
SmaI | CCC^GGG | Blocked by CpG methylation |
Csp6I | G^TAC | Not influenced by CpG methylation |
RsaI | GT^AC | Overlapping CpG methylation may influence DNA cleavage |
Ecl136II | GAG^CTC | Overlapping CpG methylation may influence DNA cleavage |
SacI | GAGCT^C | Not influenced by CpG methylation |
EcoRII | ^CC(A/T)GG | Blocked by Dcm methylation |
MvaI | CC^ (A/T)GG | Not influenced by Dcm methylation |
HpaII | C^CGG | Blocked by CpG methylation |
MspI | C^CGG | Not influenced by CpG methylation |
Overlapping methylase sequence is highlighted. m6A = N6-methyladenine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
BamHI FastDigest BamHI |
G
Gm6ATCC
| No effect |
BclI FastDigest BclI |
T
Gm6ATCA
| Blocked |
BglII FastDigest BglII |
A
Gm6ATCT
| No effect |
Bsp143I (Sau3AI) FastDigest Bsp143I | Gm6ATC | No effect |
BspPI (AlwI) |
G
Gm6ATC | Blocked |
DpnI FastDigest DpnI | Gm6ATC | Cleaves only Dam methylated DNA |
MboI FastDigest MboI | Gm6ATC | Blocked |
PsuI (BstYI) FastDigest PsuI |
R
Gm6ATCY
| No effect |
PvuI FastDigest PvuI |
C
Gm6ATCG
| No effect |
SfaAI (AsiSI) FastDigest SfaAI |
GC
Gm6ATCGC
| No effect |
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
AloI |
5'...GAAC(N)
4Gm6A TCC...3'
3'...CTTG(N) 4C Tm6AGG...5' | Not determined |
BseJI (BsaBI) FastDigest BseJI |
5'...
Gm6A TC(N)
3ATC...3'
3'... C Tm6AG(N) 3TAG...5' | Blocked |
Bsh1285I (BsiEI) FastDigest Bsh1285I |
5'...C
Gm6A TCG...3'
3'...G C Tm6AGC...5' | Impaired |
Bsp68I (NruI) |
5'...
Gm6A TCGC
Gm6A TC...3'
3'... C Tm6AGCG C Tm6AG...5' | No effect |
FastDigest RruI |
5'...
Gm6A TCGC
Gm6A TC...3'
3'... C Tm6AGCG C Tm6AG...5' | Impaired |
Bsu15I (ClaI) FastDigest Bsu15I |
5'...ATC
Gm6A TC...3'
3'...TAG C Tm6AG...5' | Blocked |
FspBI (BfaI) FastDigest FspBI |
5'...CTA
Gm6A TC...3'
3'...GAT C Tm6AG...5' | No effect |
HphI |
5'...GGT
Gm6A TC...3'
3'...CCA C Tm6AG...5' | Blocked |
Kpn2I (BspEI) FastDigest Kpn2I |
5'...TCCG
Gm6A TC...3'
3'...AGGC C Tm6AG...5' 5'... Gm6A TCCG Gm6A TC...3' 3'... C Tm6AGGC C Tm6AG...5' | No effect Not determined |
MboII FastDigest MboII |
5'...GAA
Gm6A TC...3'
3'...CTT C Tm6AG...5' | Blocked |
PagI (BspHI) |
5'...TCAT
Gm6A TC...3'
3'...AGTA C Tm6AG...5' 5'... Gm6A TCAT Gm6A TC...3' 3'... C Tm6AGTA C Tm6AG...5' | Impaired Not determined |
PfoI FastDigest PfoI |
5'...TCCNG
Gm6A TC...3'
3'...AGGNC C Tm6AG...5' | Impaired |
SmoI (SmlI) |
5'...CTYRA
Gm6A TC...3'
3'...GARYT C Tm6AG...5' | Impaired |
TaqI FastDigest TaqI |
5'...TC
Gm6A TC...3'
3'...AG C Tm6AG...5' | Blocked |
XbaI FastDigest XbaI |
5'...TCTA
Gm6A TC...3'
3'...AGAT C Tm6AG...5' | Blocked |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest CsiI |
A
Cm5CWGGT
| No effect |
EcoRII | Cm5CWGG | Blocked |
MvaI (BstNI) FastDigest MvaI | Cm6CWGG | No effect |
PasI |
C
Cm5CWGGG
| No effect |
SgeI | Cm5CWGG | Cleaves only methylated DNA |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest FokI |
5'...
Cm5CW GGATG...3'
3'... G GWm5CCTAC...5' | No effect |
Acc65I (Asp718I) FastDigest Acc65I |
5'...GGTA
Cm5CW GG...3'
3'...CCAT G GWm5CC...5' 5'... Cm5CW GGTA Cm5CW GG...3' 3'... G GWm5CCAT G GWm5CC...5' | Impaired Blocked |
AloI |
5'...GAAC(N)
6T
Cm5CW GG...3'
3'...CTTG(N) 6A G GWm5CC...5' | No effect |
ApaI FastDigest ApaI |
5'...GGGC
Cm5CW GG...3'
3'...CCCG G GWm5CC...5' | Impaired |
BamHI FastDigest BamHI |
5'...
Cm5CW GGAT
Cm5CW GG...3'
3'... G GWm5CCTA G GWm5CC...5' | No effect |
BfuI (BciVI) |
5'...GTAT
Cm5CW GG...3'
3'...CATA G GWm5CC...5' | No effect |
BglI FastDigest BglI |
5'...G
Cm5CW GGNNGGC...3'
3'...C G GWm5CCNNCCG...5' | No effect |
Bme1390I (ScrFI) FastDigest Bme1390I |
5'...
Cm5CW GG...3'
3'... G GWm5CC...5' | Blocked |
BseDI (BsaJI) FastDigest BseDI |
5'...
Cm5CW GGG...3'
3'... G GWm5CCC...5' | No effect |
BseGI (BtsCI) FastDigest BseGI |
5'...
Cm5CW GGATG...3'
3'... G GWm5CCTAC...5' | No effect |
BseLI (BslI) FastDigest BseLI |
5'...
Cm5CW GG(N)
4GG...3'
3'... G GWm5CC(N) 4CC...5' 5'... Cm5CW GGN Cm5CW GG...3' 3'... G GWm5CCN G GWm5CC...5' | Impaired Blocked |
BseSI (Bme1580I) FastDigest BseSI |
5'...GKGC
Cm5CW GG...3'
3'...CMCG G GWm5CC...5' | No effect |
BshNI (BanI) FastDigest BshNI |
5'...GGYR
Cm5CW GG...3'
3'...CCRY G GWm5CC...5' 5'... Cm5CW GGYR Cm5CW GG...3' 3'... G GWm5CCRY G GWm5CC...5' | Impaired Not determined |
Bsp120I (PspOMI) FastDigest Bsp120I |
5'...GGGC
Cm5CW GG...3'
3'...CCCG G GWm5CC...5' | Blocked |
BspLI (NlaIV) FastDigest BspLI |
5'...GGNN
Cm5CW GG...3'
3'...CCNN G GWm5CC...5' 5'... Cm5CW GGNN Cm5CW GG...3' 3'... G GWm5CCNN G GWm5CC...5' | No effect Blocked |
BspPI (AlwI) |
5'...
Cm5CW GGATC...3'
3'... G GWm5CCTAG...5' | No effect |
BstXI FastDigest BstXI |
5'...
Cm5CA GG(N)
4TGG...3'
3'... G GTm5CC(N) 4ACC...5' 5'... Cm5CA GGNN Cm5CT GG...3' 3'... G GTm5CCNN G GAm5CC...5' | No effect Blocked |
BsuRI (HaeIII) FastDigest BsuRI |
5'...GG
Cm5CW GG...3'
3'...CC G GWm5CC...5' 5'... Cm5CW GGCm5CW GG...3' 3'... G GWm5CCG GWm5CC...5' | No effect No effect |
CaiI (AlwNI) FastDigest CaiI |
5'...CAGNN
Cm5CT GG...3'
3'...GTCNN G GTm5CC...5' | Blocked |
Cfr13I (Sau96I) FastDigest Cfr13I |
5'...GGN
Cm5CW GG...3'
3'...CCN G GWm5CC...5' | Blocked |
CfrI (EaeI) |
5'...YGG
Cm5CW GG...3'
3'...RCC G GWm5CC...5' | Blocked |
Eco147I (StuI) FastDigest Eco147I |
5'...AGG
Cm5CT GG...3'
3'...TCC G GAm5CC...5' | Blocked |
Eco24I (BanII) |
5'...
Cm5CW GGGC
Cm5CW GG...3'
3'... G GWm5CCCG G GWm5CC...5' | No effect |
Eco31I (BsaI) FastDigest Eco31I |
5'...
Cm5CW GGTCTC...3'
3'... G GWm5CCAGAG...5' | Impaired |
Eco47I (AvaII) FastDigest Eco47I |
5'...GGW
Cm5CW GG...3'
3'...CCW G GWm5CC...5' | Blocked |
Eco91I (BstEII) FastDigest Eco91I |
5'...
Cm5CW GGTNA
Cm5CW GG...3'
3'... G GWm5CCANT G GWm5CC...5' | No effect |
EcoO109I (DraII) FastDigest EcoO109I |
5'...RGGN
Cm5CT GG...3'
3'...YCCN G GAm5CC...5' | Blocked |
EheI (SfoI) FastDigest EheI |
5'...
Cm5CW GGCG
Cm5CW GG...3'
3'... G GWm5CCGC G GWm5CC...5' | No effect |
FaqI (BsmFI) FastDigest FaqI |
5'...
Cm5CW GGGAC...3'
3'... G GWm5CCCTG...5' | No effect |
GsuI (BpmI) FastDigest GsuI |
5'...CT
Cm5CA GG...3'
3'...GA G GTm5CC...5' | Blocked |
Hin1I (BsaHI) FastDigest Hin1I |
5'...GRCG
Cm5CW GG...3'
3'...CYGC G GWm5CC...5' | Impaired |
HphI |
5'...
Cm5CW GGTGA...3'
3'... G GWm5CCACT...5' | No effect |
KpnI FastDigest KpnI |
5'...GGTA
Cm5CW GG...3'
3'...CCAT G GWm5CC...5' 5'... Cm5CW GGTA Cm5CW GG...3' 3'... G GWm5CCAT G GWm5CC...5' | No effect No effect |
MlsI (MscI) FastDigest MlsI |
5'...TGG
Cm5CA GG...3'
3'...ACC G GTm5CC...5' | Blocked |
PfoI FastDigest PfoI |
5'...T
Cm5CA GGA...3'
3'...A G GTm5CCT...5' | Blocked |
Psp5II (PpuMI) FastDigest Psp5II |
5'...RGGW
Cm5CT GG...3'
3'...YCCW G GAm5CC...5' | Blocked |
SduI (Bsp1286I) |
5'...
Cm5CW GGGC
Cm5CW GG...3'
3'... G GWm5CCCG G GWm5CC...5' | No effect |
SfiI FastDigest SfiI |
5'...GG
Cm5CW GGNNGGCC...3'
3'...CC G GWm5CCNNCCGG...5' 5'... Cm5CW GGCC(N) 5GG Cm5CW GG...3' 3'... G GWm5CCGG(N) 5CC G GWm5CC...5' | Impaired No effect |
SgsI (AscI) FastDigest SgsI |
5'...
Cm5CW GGCGCG
Cm5CW GG...3'
3'... G GWm5CCGCGC G GWm5CC...5' | No effect |
SspDI (KasI) |
5'…
Cm5CW GGCG
Cm5CW GG...3'
3'… G GWm5CCGC G GWm5CC...5' | No effect |
Van91I (PflMI) FastDigest Van91I |
5'...
Cm5CA GG(N)
3TGG...3'
3'... G GTm5CC(N) 3ACC...5' | Blocked |
XagI (EcoNI) FastDigest XagI |
5'...
Cm5CT GG(N)
3AGG...3'
3'... G GAm5CC(N) 3TCC...5' 5'... Cm5CT GGN Cm5CA GG...3' 3'... G GAm5CCN G GTm5CC...5' | No effect No effect |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest BmsI |
5'...GCAT
m5C G...3'
3'...CGTA Gm5C...5' 5'... m5C GGATG...3' 3'... Gm5CCTAC...5' | Impaired No effect |
FastDigest KflI |
5'...GGGWCC
m5C G...3'
3'...CCCWGG Gm5C...5' 5'...CCCAG m5C G...3' 3'...GGGTC Gm5C...5' | Blocked Impaired |
AarI |
5'...CACCTG
m5C G...3'
3'...GTGGAC Gm5C...5' | Impaired |
FastDigest AasI |
5'...
m5C GAC(N)
6GT
m5C G...3'
3'... Gm5CTG(N) 6CA Gm5C...5' | Not determined |
FastDigest AasI |
5'...GA
m5C G(N)
4m5C GTC...3'
3'...CT Gm5C(N) 4 Gm5CAG...5' | Not determined |
Acc65I (Asp718I) FastDigest Acc65I |
5'...GGTAC
m5C G...3'
3'...CCATG Gm5C...5' 5'... m5C GGTAC m5C G...3' 3'... Gm5CCATG Gm5C...5' | Impaired Blocked |
AdeI (DraIII) FastDigest AdeI |
5'...CA
m5C GNNGTG...3'
3'...GT Gm5CNNCAC...5' 5'...CA m5C GN m5C GTG...3' 3'...GT Gm5CN Gm5CAC...5' | Impaired Blocked |
AjuI |
5'...
m5C GAA(N)
7TTGG...3'
3'... Gm5CTT(N) 7AACC...5' | No effect |
AlfI |
5'...GCA(N)
6TG
m5C G...3'
3'...CGT(N) 6AC Gm5C...5' | No effect |
AloI |
5'...
m5C GAAC(N)
6TCC...3'
3'... Gm5CTTG(N) 6AGG...5' 5'...GAAC(N) 6TC m5C G...3' 3'...CTTG(N) 6AG Gm5C...5' | Blocked No effect |
AloI |
5'...GAA
m5C G(N)
5TCC...3'
3'...CTT Gm5C(N) 5AGG...5' | No effect |
Alw21I (BsiHKAI) FastDigest Alw21I |
5'...
m5C GWGCW
m5C G...3'
3'... Gm5CWCGW Gm5C...5' | No effect |
Alw26I FastDigest Alw26I |
5'...GTCT
m5C G...3'
3'...CAGA Gm5C...5' 5'... m5C GTCTC...3' 3'... Gm5CAGAG...5' | No effect Blocked |
Alw44I FastDigest Alw44I |
5'...GTGCA
m5C G...3'
3'...CACGT Gm5C...5' | Blocked |
ApaI FastDigest ApaI |
5'...GGGCC
m5C G...3'
3'...CCCGG Gm5C...5' 5'... m5C GGGCC m5C G...3' 3'... Gm5CCCGG Gm5C...5' | Impaired Impaired |
BamHI FastDigest BamHI |
5'...
m5C GGATC
m5C G...3'
3'... Gm5CCTAG Gm5C...5' | No effect |
BfuI (BciVI) |
5'...
m5C GTATCC...3'
3'... Gm5CATAGG...5' 5'...GTATC m5C G...3' 3'...CATAG Gm5C...5' | No effect No effect |
BglI FastDigest BglI |
5'...GC
m5C G(N)
3m5C GGC...3'
3'...CG Gm5C(N) 3 Gm5CCG...5' 5'...GCC(N) 5GG m5C G...3' 3'...CGG(N) 5CC Gm5C...5' | No effect Impaired |
BglI FastDigest BglI |
5'...
m5C GCC(N)
5GG
m5C G...3'
3'... Gm5CGG(N)5CC Gm5C...5' | Blocked |
Bme1390I (ScrFI) FastDigest Bme1390I |
5'...C
m5C GGG...3'
3'...G Gm5CCC...5' | Blocked |
BoxI (PshAI) FastDigest BoxI |
5'...GA
m5C G(N)
3GTC...3'
3'...CT Gm5C(N) 3CAG...5' 5'...GA m5C GNN m5C GTC...3' 3'...CT Gm5CNN Gm5CAG...5' | Impaired Blocked |
BoxI (PshAI) FastDigest BoxI |
5'...GAC(N)
4GT
m5C G...3'
3'...CTG(N) 4CA Gm5C...5' | No effect |
BpiI (BbsI) FastDigest BpiI |
5'...GAAGA
m5C G...3'
3'...CTTCT Gm5C...5' 5'... m5C GAAGAC...3' 3'... Gm5CTTCTG...5' | No effect No effect |
BplI FastDigest BplI |
5'...
m5C GAG(N)
5CT
m5C G...3'
3'... Gm5CTC(N) 5GA Gm5C...5' | No effect |
Bpu10I FastDigest Bpu10I |
5'...CCTNAG
m5C G...3'
3'...GGANTC Gm5C...5' | No effect |
Bpu1102I (BlpI) FastDigest Bpu1102I |
5'...
m5C GCTNAG
m5C G...3'
3'... Gm5CGANTC Gm5C...5' | No effect |
BseDI (BsaJI) FastDigest BseDI |
5'...C
m5C Gm5C GG...3'
3'...G Gm5C Gm5CC...5' | No effect |
BseGI (BtsCI) FastDigest BseGI |
5'...
m5C GGATG...3'
3'... Gm5CCTAC...5' | No effect |
BseJI (BsaBI) FastDigest BseJI |
5'...GAT(N)
4AT
m5C G...3'
3'...CTA(N) 4TA Gm5C...5' 5'... m5C GAT(N) 4AT m5C G...3' 3'... Gm5CTA(N) 4TA Gm5C...5' | No effect Not determined |
BseLI (BslI) FastDigest BseLI |
5'...C
m5C G(N)
6GG...3'
3'...G Gm5C(N) 6CC...5' 5'...C m5C G(N) 5m5C GG...3' 3'...G Gm5C(N) 5Gm5CC...5' | Impaired Blocked |
BseMI (BsrDI) FastDigest BseMI |
5'...
m5C GCAATG...3'
3'... Gm5CGTTAC...5' | No effect |
BseSI (Bme1580I) FastDigest BseSI |
5'...
m5C GKGCM
m5C G...3'
3'... Gm5CMCGK Gm5C...5' | No effect |
BseXI (BbvI) FastDigest BseXI |
5'...
m5C GCAG
m5C G...3'
3'... Gm5CGTC Gm5C...5' | No effect |
BshNI (BanI) FastDigest BshNI |
5'...GGYRC
m5C G...3'
3'...CCRYG Gm5C...5' 5'... m5C GGYRC m5C G...3' 3'... Gm5CCRYG Gm5C...5' | Impaired Blocked |
BshNI (BanI) FastDigest BshNI |
5'...GG
m5C GCC...3'
3'...CC Gm5CGG...5' | Impaired |
Bsp120I (PspOMI) FastDigest Bsp120I |
5'...GGGCC
m5C G...3'
3'...CCCGG Gm5C...5' | Blocked |
Bsp143I (Sau3AI) FastDigest Bsp143I |
5'...GAT
m5C G...3'
3'...CTA Gm5C...5' | Blocked |
BspLI (NlaIV) FastDigest BspLI |
5'...GGNNC
m5C G...3'
3'...CCNNG Gm5C...5' 5'... m5C GGNNC m5C G...3' 3'... Gm5CCNNG Gm5C...5' | No effect Blocked |
BspOI (BmtI) FastDigest BspOI |
5'...GCTAG
m5C G...3'
3'...CGATC Gm5C...5' 5'... m5C GCTAG m5C G...3' 3'... Gm5CGATC Gm5C...5' | No effect No effect |
BspPI (AlwI) |
5'...GGAT
m5C G...3'
3'...CCTA Gm5C...5' 5'... m5C GGATC...3' 3'... Gm5CCTAG...5' | No effect No effect |
Bst1107I (BstZ17I) FastDigest Bst1107I |
5'...GTATA
m5C G...3'
3'...CATAT Gm5C...5' 5'... m5C GTATA m5C G...3' 3'... Gm5CATAT Gm5C...5' | Impaired Blocked |
BsuRI (HaeIII) FastDigestBsuRI |
5'...
m5C GGC
m5C G...3'
3'... Gm5CCG Gm5C...5' | No effect |
BveI (BspMI) FastDigest BveI |
5'...ACCTG
m5C G...3'
3'...TGGAC Gm5C...5' | Impaired |
Cfr13I (Sau96I) FastDigest Cfr13I |
5'...GGNC
m5C G...3'
3'...CCNG Gm5C...5' | Blocked |
CfrI (EaeI)NO SKU |
5'...YGGC
m5C G...3'
3'...RCCG Gm5C...5' | Blocked |
Csp6I (CviQI) FastDigest Csp6I |
5'...
m5C GTA
m5C G...3'
3'... Gm5CAT Gm5C...5' | No effect |
DpnI FastDigest DpnI |
5'...Gm6A T
m5C G...3'
3'...C Tm6A Gm5C...5' | Cleaves only Dam methylated DNA |
Eam1104I (EarI) FastDigest Eam1104I |
5'...CTCTT
m5C G...3'
3'...GAGAA Gm5C...5' | No effect |
Eam1105I (AhdI) FastDigest Eam1105I |
5'...GA
m5C G(N)
3m5C GTC...3'
3'...CT Gm5C(N) 3 Gm5CAG...5' 5'...GAC(N) 5GT m5C G...3' 3'...CTG(N) 5CA Gm5C...5' | No effect No effect |
Eam1105I (AhdI) FastDigest Eam1105I |
5'...
m5C GAC(N)
5GT
m5C G...3'
3'... Gm5CTG(N) 5CA Gm5C...5' | Not determined |
Ecl136II (EcoICRI) FastDigest Ecl136II |
5'...GAGCT
m5C G...3'
3'...CTCGA Gm5C...5' 5'... m5C GAGCT m5C G...3' 3'... Gm5CTCGA Gm5C...5' | Impaired Blocked |
Eco24I (BanII) |
5'...
m5C GRGCY
m5C G...3'
3'... Gm5CYCGR Gm5C...5' | No effect |
Eco31I (BsaI) FastDigest Eco31I |
5'...GGTCT
m5C G...3'
3'...CCAGA Gm5C...5' 5'... m5C GGTCTC...3' 3'... Gm5CCAGAG...5' | No effect Impaired |
Eco32I (EcoRV) FastDigest Eco32I |
5'...
m5C GATAT
m5C G...3'
3'... Gm5CTATA Gm5C...5' | No effect |
Eco47I (AvaII) FastDigest Eco47I |
5'...GGWC
m5C G...3'
3'...CCWG Gm5C...5' | Blocked |
Eco91I (BstEII) FastDigest Eco91I |
5'...
m5C GGTNAC
m5C G...3'
3'... Gm5CCANTG Gm5C...5' | No effect |
EcoRI FastDigest EcoRI |
5'...
m5C GAATTm5C G...3'
3'... Gm5CTTAA Gm5C...5' | Impaired |
FaqI (BsmFI) FastDigest FaqI |
5'...GGGA
m5C G...3'
3'...CCCT Gm5C...5' 5'... m5C GGGAC...3' 3'... Gm5CCCTG...5' | Blocked Blocked |
HincII (HindII) FastDigest HincII |
5'...GTYRA
m5C G...3'
3'...CARYT Gm5C...5' 5'... m5C GTYRA m5C G...3' 3'... Gm5CARYT Gm5C...5' | Impaired Blocked |
HincII (HindII) FastDigest HincII |
5'...GT
m5C GAC...3'
3'...CA Gm5CTG...5' | Impaired |
HinfI FastDigest HinfI |
5'...GANT
m5C G...3'
3'...CTNA Gm5C...5' 5'... m5C GANT m5C G...3' 3'... Gm5CTNA Gm5C...5' | Impaired Impaired |
HphI |
5'...
m5C GGTGA...3'
3'... Gm5CCACT...5' | No effect |
Hpy8I (MjaIV) FastDigest Hpy8I |
5'...GTNNA
m5C G...3'
3'...CANNT Gm5C...5' 5'... m5C GTNNA m5C G...3' 3'... Gm5CANNT Gm5C...5' | Impaired Not determined |
HpyF10VI (MwoI) FastDigest HpyF10VI |
5'...GC(N)
7G
m5C G...3'
3'...CG(N) 7C Gm5C...5' 5'... m5C GC(N) 7G m5C G...3' 3'... Gm5CG(N) 7C Gm5C...5' | Impaired Blocked |
HpyF10VI (MwoI) FastDigest HpyF10VI |
5'...G
m5C G(N)
6GC...3'
3'...C Gm5C(N) 6CG...5' 5'...G m5C G(N) 5m5C GC...3' 3'...C Gm5C(N) 5 Gm5CG...5' | Impaired Not determined |
KpnI FastDigest KpnI |
5'...
m5C GGTAC
m5C G...3'
3'... Gm5CCATG Gm5C...5' | No effect |
KspAI (HpaI) FastDigest KspAI |
5'...GTTAA
m5C G...3'
3'...CAATT Gm5C...5' 5'... m5C GTTAA m5C G...3' 3'... Gm5CAATT Gm5C...5' | Impaired Blocked |
LguI (SapI) FastDigest LguI |
5'...
m5C GCTCTTC...3'
3'... Gm5CGAGAAG...5' 5'...GCTCTT m5C G...3' 3'...CGAGAA Gm5C...5' | No effect No effect |
Lsp1109I (BbvI) FastDigest Lsp1109I |
5'...
m5C GCAG
m5C G...3'
3'... Gm5CGTC Gm5C...5' | No effect |
LweI (SfaNI) |
5'...GCAT
m5C G...3'
3'...CGTA Gm5C...5' | Impaired |
MbiI (BsrBI) FastDigest MbiI |
5'...
m5C GAGCGG...3'
3'... Gm5CTCGCC...5' | Blocked |
MboI FastDigest MboI |
5'...
m5C GAT
m5C G...3'
3'... Gm5CTA Gm5C...5' | No effect |
MboII FastDigest MboII |
5'...
m5C GAAGA...3'
3'... Gm5CTTCT...5' | No effect |
MnlI FastDigest MnlI |
5'...CCT
m5C G...3'
3'...GGA Gm5C...5' | No effect |
MssI (PmeI) FastDigest MssI |
5'...GTTTAAA
m5C G...3'
3'...CAAATTT Gm5C...5' 5'... m5C GTTTAAA m5C G...3' 3'... Gm5CAAATTT Gm5C...5' | No effect No effect |
Mva1269I (BsmI) FastDigest Mva1269I |
5'...GAATG
m5C G...3'
3'...CTTAC Gm5C...5' 5'... m5C GAATGC...3' 3'... Gm5CTTACG...5' | No effect No effect |
NheI FastDigest NheI |
5'...GCTAG
m5C G...3'
3'...CGATC Gm5C...5' 5'... m5C GCTAG m5C G...3' 3'... Gm5CGATC Gm5C...5' | Impaired Blocked |
NmuCI (Tsp45I) FastDigest NmuCI |
5'...GTCA
m5C G...3'
3'...CAGT Gm5C...5' 5'... m5C GTCA m5C G...3' 3'... Gm5CAGT Gm5C...5' | Impaired Impaired |
OliI (AleI) FastDigest OliI |
5'...CA
m5C GNN
m5C GTG...3'
3'...GT Gm5CNN Gm5CAC...5' 5'...CA m5C GNNNGTG...3' 3'...GT Gm5CNNNCAC...5' | Impaired No effect |
PaeI (SphI) FastDigest PaeI |
5'...
m5C GCATG
m5C G...3'
3'... Gm5CGTAC Gm5C...5' | No effect |
PdmI (XmnI) FastDigest PdmI |
5'...GAA(N)
4TT
m5C G...3'
3'...CTT(N) 4AA Gm5C...5' 5'... m5C GAA(N) 4TT m5C G...3' 3'... Gm5CTT(N) 4AA Gm5C...5' | Impaired Blocked |
PfeI (TfiI) FastDigest PfeI |
5'...GAWT
m5C G...3'
3'...CTWA Gm5C...5' | Blocked |
PfoI FastDigest PfoI |
5'...TC
m5C GGGA...3'
3'...AG Gm5CCCT...5' | Blocked |
FastDigest PspFI | 5'...CCCAGm5C G...3' | Impaired |
PsuI (BstYI) FastDigest PsuI |
5'...
m5C GGATC
m5C G...3'
3'... Gm5CCTAG Gm5C...5' | No effect |
PsyI (Tth111I) FastDigest PsyI |
5'...GA
m5C GNNGT
m5C G...3'
3'...CT Gm5CNNCA Gm5C...5' | No effect |
RsaI FastDigest RsaI |
5'...GTA
m5C G...3'
3'...CAT Gm5C...5' 5'... m5C GTA m5C G...3' 3'... Gm5CAT Gm5C...5' | Impaired Blocked |
RseI (MslI) FastDigest RseI |
5'...CA
m5C GNNNRTG...3'
3'...GT Gm5CNNNYAC...5' | No effect |
SacI FastDigest SacI |
5'...
m5C GAGCT
m5C G...3'
3'... Gm5CTCGA Gm5C...5' | No effect |
SatI (Fnu4HI) FastDigest SatI |
5'...G
m5C GGC...3'
3'...C Gm5CCG...5' 5'...GCNG m5C G...3' 3'...CGNC Gm5C...5' | Blocked Blocked |
SchI (MlyI) FastDigest SchI |
5'...GAGT
m5C G...3'
3'...CTCA Gm5C...5' 5'... m5C GAGTC...3' 3'... Gm5CTCAG...5' | No effect No effect |
SduI (Bsp1286I) |
5'...
m5C GDGCH
m5C G...3'
3'... Gm5CHCGD Gm5C...5' | No effect |
SfiI FastDigest SfiI |
5'...
m5C GGCC(N)
5GGC
m5C G...3'
3'... Gm5CCGG(N) 5CCG Gm5C...5' 5'...GGC m5C G(N) 4GGCC...3' 3'...CCG Gm5C(N) 4CCGG...5' | No effect Impaired |
SfiI FastDigest SfiI |
5'...GGC
m5C G(N)
3m5C GGCC...3'
3'...CCG Gm5C(N) 3 Gm5CCGG...5' | Blocked |
SgeI |
5'...
m5C GNG...3'
3'... Gm5CNC...5' 5'... m5C Gm5 C G...3' 3'... Gm5CGm5C...5' | Cleaves only methylated DNA Cleaves only methylated DNA |
SmoI (SmlI) |
5'...CT
m5C GGT...3'
3'...GA Gm5CCA...5' | No effect |
TaaI (HpyCH4III) FastDigest TaaI |
5'...A
m5C GGT...3'
3'...T Gm5CCA...5' | Impaired |
XapI (ApoI) |
5'...
m5C GAATT
m5C G...3'
3'... Gm5CTTAA Gm5C...5' | No effect |
XceI (NspI) FastDigest XceI |
5'...RCATG
m5C G...3'
3'...YGTAC Gm5C...5' 5'... m5C GCATG m5C G...3' 3'... Gm5CGTAC Gm5C...5' | No effect Not determined |
XmiI (AccI) FastDigest XmiI |
5'...GT
m5C GAC...3'
3'...CA Gm5CTG...5' 5'...GTMKA m5C G...3' 3'...CAKMT Gm5C...5' | Blocked Blocked |
m6A = N6-methyladenine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
AatII FastDigest AatII |
5'...TGm6ACGTC(N)
4 TGCT...3'
3'...AC TGCAG(N) 4m6ACGA...5' | No effect |
AluI FastDigest AluI |
5'...TGm6AGCT(N)
5 TGCT...3'
3'...AC TCGA(N) 5m6ACGA...5' | Blocked |
BclI FastDigest BclI |
5'...TGm6A TCA(N)
5 TGCT...3'
3'...AC Tm6AGT(N) 5m6ACGA...5' | Blocked |
BglII FastDigest BglII |
5'...TGm6AGATCT(N)
3 TGCT...3'
3'...AC TCTAGA(N) 3m6ACGA...5' | Impaired |
Bpu10I FastDigest Bpu10I |
5'...CCTGm6AGC(N)
6 TGCT...3'
3'...GGAC TCG(N) 6m6ACGA...5' | No effect |
Bsp143I (Sau3AI) FastDigest Bsp143I |
5'...TGm6ATC(N)
6 TGCT...3'
3'...AC TAG(N) 6m6ACGA...5' | No effect |
EcoRI FastDigest EcoRI |
5'...TGm6AATTC(N)
4 TGCT...3'
3'...AC TTAAG(N) 4m6ACGA...5' | No effect |
HincII (HindII) FastDigest HincII |
5'...GTTGm6AC(N)
7 TGCT...3'
3'...CAAC TG(N) 7m6ACGA...5' | Blocked |
HindIII FastDigest HindIII |
5'...TGm6AAGCTT(N)
3 TGCT...3'
3'...AC TTCGAA(N) 3m6ACGA...5' | Impaired |
HinfI FastDigest HinfI |
5'...TGm6ANTC(N)
5 TGCT...3'
3'...AC TNAG(N) 5m6ACGA...5' | Blocked |
HphI |
5'...GGTGm6A(N)
8 TGCT...3'
3'...CCAC T(N) 8m6ACGA...5' | Blocked |
MboI FastDigest MboI |
5'...TGm6ATC(N)
6 TGCT...3'
3'...AC TAG(N) 6m6ACGA...5' | Blocked |
MboII FastDigest MboII |
5'...TGm6AAGA(N)
5 TGCT...3'
3'...AC TTCT(N) 5m6ACGA...5' | No effect |
MluI FastDigest MluI |
5'...TGm6ACGCGT(N)
3 TGCT...3'
3'...AC TGCGCA(N) 3m6ACGA...5' | No effect |
MnlI FastDigest MnlI |
5'...TGm6AGG(N)
6 TGCT...3'
3'...AC TCC(N) 6m6ACGA...5' | Blocked |
NdeI FastDigest NdeI |
5'...TGm6A(N)
4CATA TGCT...3'
3'...AC T(N) 4GTATm6ACGA...5' | No effect |
OliI (AleI) FastDigest OliI |
5'...TGm6A(N)
4CACG TGCT...3'
3'...AC T(N) 4GTGCm6ACGA...5' | Blocked |
PaeI (SphI) FastDigest PaeI |
5'...TGm6A(N)
5GCA TGCT...3'
3'...AC T(N) 5CGTm6ACGA...5' | Blocked |
PagI (BspHI) |
5'...TCATGm6A(N)
8 TGCT...3'
3'...AGTAC T(N) 8m6ACGA...5' | Impaired |
SacI FastDigest SacI |
5'...TGm6AGCTC(N)
4 TGCT...3'
3'...AC TCGAG(N) 4m6ACGA...5' | No effect |
ScaI FastDigest ScaI |
5'...TGm6AGTACT(N)
3 TGCT...3'
3'...AC TCATGA(N) 3m6ACGA...5' | Blocked |
SspI FastDigest SspI |
5'...TGm6AATATT(N)
3 TGCT...3'
3'...AC TTATAA(N) 3m6ACGA...5' | No effect |
TasI (Tsp509I) FastDigest TTasI |
5'...TGm6AATT(N)
5 TGCT...3'
3'...AC TTAA(N) 5m6ACGA...5' | Blocked |
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
Alw21I (BsiHKAI) FastDigest Alw21I |
5'...Am6AC(N)
6G TGCWC...3'
3'...T TG(N) 6Cm6ACGWG...5' | No effect |
Alw44I FastDigest Alw44I |
5'...Am6AC(N)
6G TGCAC...3'
3'...T TG(N) 6Cm6ACGTG...5' | No effect |
BseSI (Bme1580I) FastDigest BseSI |
5'...Am6AC(N)
6G TGCMC...3'
3'...T TG(N) 6Cm6ACGKG...5' | Impaired |
DraI FastDigest DraI |
5'...TTTAAm6AC(N)
6G TGC...3'
3'...AAATT TG(N) 6Cm6ACG...5' | Blocked |
HincII (HindII) FastDigest HincII |
5'...GTYAm6AC(N)
6G TGC...3'
3'...CART TG(N) 6Cm6ACG...5' | Blocked |
KspAI (HpaI) FastDigest KspAI |
5'...GTTAm6AC(N)
6G TGC...3'
3'...CAAT TG(N) 6Cm6ACG...5' | Blocked |
MluI FastDigest MluI |
5'...Am6ACGCGTNNG TGC...3'
3'...T TGCGCANNCm6ACG...5' | No effect |
MssI (PmeI) FastDigest MssI |
5'...GTTTAAm6AC(N)
6G TGC...3'
3'...CAAATT TG(N) 6Cm6ACG...5' | Blocked |
NsbI (FspI) FastDigest NsbI |
5'...Am6AC(N)
6G TGCGCA...3'
3'...T TG(N) 6Cm6ACGCGT...5' | No effect |
OliI (AleI) FastDigest OliI |
5'...Am6ACAC(N)
4G TGC...3'
3'...T TGTG(N) 4Cm6ACG...5' | Blocked |
RseI (MslI) FastDigest RseI |
5'...GCm6ACNNNNRTG TT...3'
3'...CG TGNNNNYACm6AT...5' | Blocked |
Tru1I (MseI) FastDigest Tru1I |
5'...TTAm6AC(N)
6G TGC...3'
3'...AAT TG(N) 6Cm6ACG...5' | Blocked |
The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases – Dam and Dcm (3, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (5, 6). The methylase encoded by the dcm gene methylates the C5-position of the internal cytosine residue within the CCWGG sequence (4, 7). To cleave with a restriction enzyme which is sensitive to the Dam or Dcm methylation, DNA should be purified from dam-, dcm- E.coli strains respectively. Control digestions should be performed with Lambda DNA (dam-, dcm-), #SD0021.
In addition to Dam and Dcm methylases, laboratory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8TGCT for EcoBI (3, 4).
DNA from higher eukaryotic organisms possesses modified 5-methylcytosine residues within CpG or CpNpG contexts (2, 8-10). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation.
Most restriction enzymes are sensitive to DNA methylation. In cases where a restriction enzyme target site overlaps a methylation site, the following digestion results are possible:
The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation. For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (11), while the isoschizomer Bsp143I is insensitive to Dam methylation. EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (12). Thus, to achieve effective DNA digestion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction enzyme to that type of methylation.
All restriction enzymes produced by Thermo Scientific have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA.
Enzyme couple | Recognition and cleavage sites | Sensitivity to methylation |
---|---|---|
Acc65I | G^GTACC | Overlapping Dcm or CpG methylation may influence DNA cleavage |
KpnI | GGTAC^C | Not influenced by Dcm or CpG methylation |
ApaI | GGGCC^C | Overlapping Dcm or CpG methylation may influence DNA cleavage |
Bsp120I | G^GGCCC | Blocked by overlapping Dcm or CpG methylation |
Bsp143I | ^GATC | Not influenced by dam, blocked by CpG methylation |
MboI | ^GATC | Blocked by Dam methylated DNA |
DpnI | GA^TC | Cleaves only Dam methylated DNA |
BspOI | GCTAG^C | Not influenced by CpG methylation |
NheI | G^CTAGC | Overlapping CpG methylation may influence DNA cleavage |
Cfr9I | C^CCGGG | CpG methylation may influence DNA cleavage |
SmaI | CCC^GGG | Blocked by CpG methylation |
Csp6I | G^TAC | Not influenced by CpG methylation |
RsaI | GT^AC | Overlapping CpG methylation may influence DNA cleavage |
Ecl136II | GAG^CTC | Overlapping CpG methylation may influence DNA cleavage |
SacI | GAGCT^C | Not influenced by CpG methylation |
EcoRII | ^CC(A/T)GG | Blocked by Dcm methylation |
MvaI | CC^ (A/T)GG | Not influenced by Dcm methylation |
HpaII | C^CGG | Blocked by CpG methylation |
MspI | C^CGG | Not influenced by CpG methylation |
Overlapping methylase sequence is highlighted. m6A = N6-methyladenine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
BamHI FastDigest BamHI |
G
Gm6ATCC
| No effect |
BclI FastDigest BclI |
T
Gm6ATCA
| Blocked |
BglII FastDigest BglII |
A
Gm6ATCT
| No effect |
Bsp143I (Sau3AI) FastDigest Bsp143I | Gm6ATC | No effect |
BspPI (AlwI) |
G
Gm6ATC | Blocked |
DpnI FastDigest DpnI | Gm6ATC | Cleaves only Dam methylated DNA |
MboI FastDigest MboI | Gm6ATC | Blocked |
PsuI (BstYI) FastDigest PsuI |
R
Gm6ATCY
| No effect |
PvuI FastDigest PvuI |
C
Gm6ATCG
| No effect |
SfaAI (AsiSI) FastDigest SfaAI |
GC
Gm6ATCGC
| No effect |
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
AloI |
5'...GAAC(N)
4Gm6A TCC...3'
3'...CTTG(N) 4C Tm6AGG...5' | Not determined |
BseJI (BsaBI) FastDigest BseJI |
5'...
Gm6A TC(N)
3ATC...3'
3'... C Tm6AG(N) 3TAG...5' | Blocked |
Bsh1285I (BsiEI) FastDigest Bsh1285I |
5'...C
Gm6A TCG...3'
3'...G C Tm6AGC...5' | Impaired |
Bsp68I (NruI) |
5'...
Gm6A TCGC
Gm6A TC...3'
3'... C Tm6AGCG C Tm6AG...5' | No effect |
FastDigest RruI |
5'...
Gm6A TCGC
Gm6A TC...3'
3'... C Tm6AGCG C Tm6AG...5' | Impaired |
Bsu15I (ClaI) FastDigest Bsu15I |
5'...ATC
Gm6A TC...3'
3'...TAG C Tm6AG...5' | Blocked |
FspBI (BfaI) FastDigest FspBI |
5'...CTA
Gm6A TC...3'
3'...GAT C Tm6AG...5' | No effect |
HphI |
5'...GGT
Gm6A TC...3'
3'...CCA C Tm6AG...5' | Blocked |
Kpn2I (BspEI) FastDigest Kpn2I |
5'...TCCG
Gm6A TC...3'
3'...AGGC C Tm6AG...5' 5'... Gm6A TCCG Gm6A TC...3' 3'... C Tm6AGGC C Tm6AG...5' | No effect Not determined |
MboII FastDigest MboII |
5'...GAA
Gm6A TC...3'
3'...CTT C Tm6AG...5' | Blocked |
PagI (BspHI) |
5'...TCAT
Gm6A TC...3'
3'...AGTA C Tm6AG...5' 5'... Gm6A TCAT Gm6A TC...3' 3'... C Tm6AGTA C Tm6AG...5' | Impaired Not determined |
PfoI FastDigest PfoI |
5'...TCCNG
Gm6A TC...3'
3'...AGGNC C Tm6AG...5' | Impaired |
SmoI (SmlI) |
5'...CTYRA
Gm6A TC...3'
3'...GARYT C Tm6AG...5' | Impaired |
TaqI FastDigest TaqI |
5'...TC
Gm6A TC...3'
3'...AG C Tm6AG...5' | Blocked |
XbaI FastDigest XbaI |
5'...TCTA
Gm6A TC...3'
3'...AGAT C Tm6AG...5' | Blocked |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest CsiI |
A
Cm5CWGGT
| No effect |
EcoRII | Cm5CWGG | Blocked |
MvaI (BstNI) FastDigest MvaI | Cm6CWGG | No effect |
PasI |
C
Cm5CWGGG
| No effect |
SgeI | Cm5CWGG | Cleaves only methylated DNA |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest FokI |
5'...
Cm5CW GGATG...3'
3'... G GWm5CCTAC...5' | No effect |
Acc65I (Asp718I) FastDigest Acc65I |
5'...GGTA
Cm5CW GG...3'
3'...CCAT G GWm5CC...5' 5'... Cm5CW GGTA Cm5CW GG...3' 3'... G GWm5CCAT G GWm5CC...5' | Impaired Blocked |
AloI |
5'...GAAC(N)
6T
Cm5CW GG...3'
3'...CTTG(N) 6A G GWm5CC...5' | No effect |
ApaI FastDigest ApaI |
5'...GGGC
Cm5CW GG...3'
3'...CCCG G GWm5CC...5' | Impaired |
BamHI FastDigest BamHI |
5'...
Cm5CW GGAT
Cm5CW GG...3'
3'... G GWm5CCTA G GWm5CC...5' | No effect |
BfuI (BciVI) |
5'...GTAT
Cm5CW GG...3'
3'...CATA G GWm5CC...5' | No effect |
BglI FastDigest BglI |
5'...G
Cm5CW GGNNGGC...3'
3'...C G GWm5CCNNCCG...5' | No effect |
Bme1390I (ScrFI) FastDigest Bme1390I |
5'...
Cm5CW GG...3'
3'... G GWm5CC...5' | Blocked |
BseDI (BsaJI) FastDigest BseDI |
5'...
Cm5CW GGG...3'
3'... G GWm5CCC...5' | No effect |
BseGI (BtsCI) FastDigest BseGI |
5'...
Cm5CW GGATG...3'
3'... G GWm5CCTAC...5' | No effect |
BseLI (BslI) FastDigest BseLI |
5'...
Cm5CW GG(N)
4GG...3'
3'... G GWm5CC(N) 4CC...5' 5'... Cm5CW GGN Cm5CW GG...3' 3'... G GWm5CCN G GWm5CC...5' | Impaired Blocked |
BseSI (Bme1580I) FastDigest BseSI |
5'...GKGC
Cm5CW GG...3'
3'...CMCG G GWm5CC...5' | No effect |
BshNI (BanI) FastDigest BshNI |
5'...GGYR
Cm5CW GG...3'
3'...CCRY G GWm5CC...5' 5'... Cm5CW GGYR Cm5CW GG...3' 3'... G GWm5CCRY G GWm5CC...5' | Impaired Not determined |
Bsp120I (PspOMI) FastDigest Bsp120I |
5'...GGGC
Cm5CW GG...3'
3'...CCCG G GWm5CC...5' | Blocked |
BspLI (NlaIV) FastDigest BspLI |
5'...GGNN
Cm5CW GG...3'
3'...CCNN G GWm5CC...5' 5'... Cm5CW GGNN Cm5CW GG...3' 3'... G GWm5CCNN G GWm5CC...5' | No effect Blocked |
BspPI (AlwI) |
5'...
Cm5CW GGATC...3'
3'... G GWm5CCTAG...5' | No effect |
BstXI FastDigest BstXI |
5'...
Cm5CA GG(N)
4TGG...3'
3'... G GTm5CC(N) 4ACC...5' 5'... Cm5CA GGNN Cm5CT GG...3' 3'... G GTm5CCNN G GAm5CC...5' | No effect Blocked |
BsuRI (HaeIII) FastDigest BsuRI |
5'...GG
Cm5CW GG...3'
3'...CC G GWm5CC...5' 5'... Cm5CW GGCm5CW GG...3' 3'... G GWm5CCG GWm5CC...5' | No effect No effect |
CaiI (AlwNI) FastDigest CaiI |
5'...CAGNN
Cm5CT GG...3'
3'...GTCNN G GTm5CC...5' | Blocked |
Cfr13I (Sau96I) FastDigest Cfr13I |
5'...GGN
Cm5CW GG...3'
3'...CCN G GWm5CC...5' | Blocked |
CfrI (EaeI) |
5'...YGG
Cm5CW GG...3'
3'...RCC G GWm5CC...5' | Blocked |
Eco147I (StuI) FastDigest Eco147I |
5'...AGG
Cm5CT GG...3'
3'...TCC G GAm5CC...5' | Blocked |
Eco24I (BanII) |
5'...
Cm5CW GGGC
Cm5CW GG...3'
3'... G GWm5CCCG G GWm5CC...5' | No effect |
Eco31I (BsaI) FastDigest Eco31I |
5'...
Cm5CW GGTCTC...3'
3'... G GWm5CCAGAG...5' | Impaired |
Eco47I (AvaII) FastDigest Eco47I |
5'...GGW
Cm5CW GG...3'
3'...CCW G GWm5CC...5' | Blocked |
Eco91I (BstEII) FastDigest Eco91I |
5'...
Cm5CW GGTNA
Cm5CW GG...3'
3'... G GWm5CCANT G GWm5CC...5' | No effect |
EcoO109I (DraII) FastDigest EcoO109I |
5'...RGGN
Cm5CT GG...3'
3'...YCCN G GAm5CC...5' | Blocked |
EheI (SfoI) FastDigest EheI |
5'...
Cm5CW GGCG
Cm5CW GG...3'
3'... G GWm5CCGC G GWm5CC...5' | No effect |
FaqI (BsmFI) FastDigest FaqI |
5'...
Cm5CW GGGAC...3'
3'... G GWm5CCCTG...5' | No effect |
GsuI (BpmI) FastDigest GsuI |
5'...CT
Cm5CA GG...3'
3'...GA G GTm5CC...5' | Blocked |
Hin1I (BsaHI) FastDigest Hin1I |
5'...GRCG
Cm5CW GG...3'
3'...CYGC G GWm5CC...5' | Impaired |
HphI |
5'...
Cm5CW GGTGA...3'
3'... G GWm5CCACT...5' | No effect |
KpnI FastDigest KpnI |
5'...GGTA
Cm5CW GG...3'
3'...CCAT G GWm5CC...5' 5'... Cm5CW GGTA Cm5CW GG...3' 3'... G GWm5CCAT G GWm5CC...5' | No effect No effect |
MlsI (MscI) FastDigest MlsI |
5'...TGG
Cm5CA GG...3'
3'...ACC G GTm5CC...5' | Blocked |
PfoI FastDigest PfoI |
5'...T
Cm5CA GGA...3'
3'...A G GTm5CCT...5' | Blocked |
Psp5II (PpuMI) FastDigest Psp5II |
5'...RGGW
Cm5CT GG...3'
3'...YCCW G GAm5CC...5' | Blocked |
SduI (Bsp1286I) |
5'...
Cm5CW GGGC
Cm5CW GG...3'
3'... G GWm5CCCG G GWm5CC...5' | No effect |
SfiI FastDigest SfiI |
5'...GG
Cm5CW GGNNGGCC...3'
3'...CC G GWm5CCNNCCGG...5' 5'... Cm5CW GGCC(N) 5GG Cm5CW GG...3' 3'... G GWm5CCGG(N) 5CC G GWm5CC...5' | Impaired No effect |
SgsI (AscI) FastDigest SgsI |
5'...
Cm5CW GGCGCG
Cm5CW GG...3'
3'... G GWm5CCGCGC G GWm5CC...5' | No effect |
SspDI (KasI) |
5'…
Cm5CW GGCG
Cm5CW GG...3'
3'… G GWm5CCGC G GWm5CC...5' | No effect |
Van91I (PflMI) FastDigest Van91I |
5'...
Cm5CA GG(N)
3TGG...3'
3'... G GTm5CC(N) 3ACC...5' | Blocked |
XagI (EcoNI) FastDigest XagI |
5'...
Cm5CT GG(N)
3AGG...3'
3'... G GAm5CC(N) 3TCC...5' 5'... Cm5CT GGN Cm5CA GG...3' 3'... G GAm5CCN G GTm5CC...5' | No effect No effect |
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
FastDigest BmsI |
5'...GCAT
m5C G...3'
3'...CGTA Gm5C...5' 5'... m5C GGATG...3' 3'... Gm5CCTAC...5' | Impaired No effect |
FastDigest KflI |
5'...GGGWCC
m5C G...3'
3'...CCCWGG Gm5C...5' 5'...CCCAG m5C G...3' 3'...GGGTC Gm5C...5' | Blocked Impaired |
AarI |
5'...CACCTG
m5C G...3'
3'...GTGGAC Gm5C...5' | Impaired |
FastDigest AasI |
5'...
m5C GAC(N)
6GT
m5C G...3'
3'... Gm5CTG(N) 6CA Gm5C...5' | Not determined |
FastDigest AasI |
5'...GA
m5C G(N)
4m5C GTC...3'
3'...CT Gm5C(N) 4 Gm5CAG...5' | Not determined |
Acc65I (Asp718I) FastDigest Acc65I |
5'...GGTAC
m5C G...3'
3'...CCATG Gm5C...5' 5'... m5C GGTAC m5C G...3' 3'... Gm5CCATG Gm5C...5' | Impaired Blocked |
AdeI (DraIII) FastDigest AdeI |
5'...CA
m5C GNNGTG...3'
3'...GT Gm5CNNCAC...5' 5'...CA m5C GN m5C GTG...3' 3'...GT Gm5CN Gm5CAC...5' | Impaired Blocked |
AjuI |
5'...
m5C GAA(N)
7TTGG...3'
3'... Gm5CTT(N) 7AACC...5' | No effect |
AlfI |
5'...GCA(N)
6TG
m5C G...3'
3'...CGT(N) 6AC Gm5C...5' | No effect |
AloI |
5'...
m5C GAAC(N)
6TCC...3'
3'... Gm5CTTG(N) 6AGG...5' 5'...GAAC(N) 6TC m5C G...3' 3'...CTTG(N) 6AG Gm5C...5' | Blocked No effect |
AloI |
5'...GAA
m5C G(N)
5TCC...3'
3'...CTT Gm5C(N) 5AGG...5' | No effect |
Alw21I (BsiHKAI) FastDigest Alw21I |
5'...
m5C GWGCW
m5C G...3'
3'... Gm5CWCGW Gm5C...5' | No effect |
Alw26I FastDigest Alw26I |
5'...GTCT
m5C G...3'
3'...CAGA Gm5C...5' 5'... m5C GTCTC...3' 3'... Gm5CAGAG...5' | No effect Blocked |
Alw44I FastDigest Alw44I |
5'...GTGCA
m5C G...3'
3'...CACGT Gm5C...5' | Blocked |
ApaI FastDigest ApaI |
5'...GGGCC
m5C G...3'
3'...CCCGG Gm5C...5' 5'... m5C GGGCC m5C G...3' 3'... Gm5CCCGG Gm5C...5' | Impaired Impaired |
BamHI FastDigest BamHI |
5'...
m5C GGATC
m5C G...3'
3'... Gm5CCTAG Gm5C...5' | No effect |
BfuI (BciVI) |
5'...
m5C GTATCC...3'
3'... Gm5CATAGG...5' 5'...GTATC m5C G...3' 3'...CATAG Gm5C...5' | No effect No effect |
BglI FastDigest BglI |
5'...GC
m5C G(N)
3m5C GGC...3'
3'...CG Gm5C(N) 3 Gm5CCG...5' 5'...GCC(N) 5GG m5C G...3' 3'...CGG(N) 5CC Gm5C...5' | No effect Impaired |
BglI FastDigest BglI |
5'...
m5C GCC(N)
5GG
m5C G...3'
3'... Gm5CGG(N)5CC Gm5C...5' | Blocked |
Bme1390I (ScrFI) FastDigest Bme1390I |
5'...C
m5C GGG...3'
3'...G Gm5CCC...5' | Blocked |
BoxI (PshAI) FastDigest BoxI |
5'...GA
m5C G(N)
3GTC...3'
3'...CT Gm5C(N) 3CAG...5' 5'...GA m5C GNN m5C GTC...3' 3'...CT Gm5CNN Gm5CAG...5' | Impaired Blocked |
BoxI (PshAI) FastDigest BoxI |
5'...GAC(N)
4GT
m5C G...3'
3'...CTG(N) 4CA Gm5C...5' | No effect |
BpiI (BbsI) FastDigest BpiI |
5'...GAAGA
m5C G...3'
3'...CTTCT Gm5C...5' 5'... m5C GAAGAC...3' 3'... Gm5CTTCTG...5' | No effect No effect |
BplI FastDigest BplI |
5'...
m5C GAG(N)
5CT
m5C G...3'
3'... Gm5CTC(N) 5GA Gm5C...5' | No effect |
Bpu10I FastDigest Bpu10I |
5'...CCTNAG
m5C G...3'
3'...GGANTC Gm5C...5' | No effect |
Bpu1102I (BlpI) FastDigest Bpu1102I |
5'...
m5C GCTNAG
m5C G...3'
3'... Gm5CGANTC Gm5C...5' | No effect |
BseDI (BsaJI) FastDigest BseDI |
5'...C
m5C Gm5C GG...3'
3'...G Gm5C Gm5CC...5' | No effect |
BseGI (BtsCI) FastDigest BseGI |
5'...
m5C GGATG...3'
3'... Gm5CCTAC...5' | No effect |
BseJI (BsaBI) FastDigest BseJI |
5'...GAT(N)
4AT
m5C G...3'
3'...CTA(N) 4TA Gm5C...5' 5'... m5C GAT(N) 4AT m5C G...3' 3'... Gm5CTA(N) 4TA Gm5C...5' | No effect Not determined |
BseLI (BslI) FastDigest BseLI |
5'...C
m5C G(N)
6GG...3'
3'...G Gm5C(N) 6CC...5' 5'...C m5C G(N) 5m5C GG...3' 3'...G Gm5C(N) 5Gm5CC...5' | Impaired Blocked |
BseMI (BsrDI) FastDigest BseMI |
5'...
m5C GCAATG...3'
3'... Gm5CGTTAC...5' | No effect |
BseSI (Bme1580I) FastDigest BseSI |
5'...
m5C GKGCM
m5C G...3'
3'... Gm5CMCGK Gm5C...5' | No effect |
BseXI (BbvI) FastDigest BseXI |
5'...
m5C GCAG
m5C G...3'
3'... Gm5CGTC Gm5C...5' | No effect |
BshNI (BanI) FastDigest BshNI |
5'...GGYRC
m5C G...3'
3'...CCRYG Gm5C...5' 5'... m5C GGYRC m5C G...3' 3'... Gm5CCRYG Gm5C...5' | Impaired Blocked |
BshNI (BanI) FastDigest BshNI |
5'...GG
m5C GCC...3'
3'...CC Gm5CGG...5' | Impaired |
Bsp120I (PspOMI) FastDigest Bsp120I |
5'...GGGCC
m5C G...3'
3'...CCCGG Gm5C...5' | Blocked |
Bsp143I (Sau3AI) FastDigest Bsp143I |
5'...GAT
m5C G...3'
3'...CTA Gm5C...5' | Blocked |
BspLI (NlaIV) FastDigest BspLI |
5'...GGNNC
m5C G...3'
3'...CCNNG Gm5C...5' 5'... m5C GGNNC m5C G...3' 3'... Gm5CCNNG Gm5C...5' | No effect Blocked |
BspOI (BmtI) FastDigest BspOI |
5'...GCTAG
m5C G...3'
3'...CGATC Gm5C...5' 5'... m5C GCTAG m5C G...3' 3'... Gm5CGATC Gm5C...5' | No effect No effect |
BspPI (AlwI) |
5'...GGAT
m5C G...3'
3'...CCTA Gm5C...5' 5'... m5C GGATC...3' 3'... Gm5CCTAG...5' | No effect No effect |
Bst1107I (BstZ17I) FastDigest Bst1107I |
5'...GTATA
m5C G...3'
3'...CATAT Gm5C...5' 5'... m5C GTATA m5C G...3' 3'... Gm5CATAT Gm5C...5' | Impaired Blocked |
BsuRI (HaeIII) FastDigestBsuRI |
5'...
m5C GGC
m5C G...3'
3'... Gm5CCG Gm5C...5' | No effect |
BveI (BspMI) FastDigest BveI |
5'...ACCTG
m5C G...3'
3'...TGGAC Gm5C...5' | Impaired |
Cfr13I (Sau96I) FastDigest Cfr13I |
5'...GGNC
m5C G...3'
3'...CCNG Gm5C...5' | Blocked |
CfrI (EaeI)NO SKU |
5'...YGGC
m5C G...3'
3'...RCCG Gm5C...5' | Blocked |
Csp6I (CviQI) FastDigest Csp6I |
5'...
m5C GTA
m5C G...3'
3'... Gm5CAT Gm5C...5' | No effect |
DpnI FastDigest DpnI |
5'...Gm6A T
m5C G...3'
3'...C Tm6A Gm5C...5' | Cleaves only Dam methylated DNA |
Eam1104I (EarI) FastDigest Eam1104I |
5'...CTCTT
m5C G...3'
3'...GAGAA Gm5C...5' | No effect |
Eam1105I (AhdI) FastDigest Eam1105I |
5'...GA
m5C G(N)
3m5C GTC...3'
3'...CT Gm5C(N) 3 Gm5CAG...5' 5'...GAC(N) 5GT m5C G...3' 3'...CTG(N) 5CA Gm5C...5' | No effect No effect |
Eam1105I (AhdI) FastDigest Eam1105I |
5'...
m5C GAC(N)
5GT
m5C G...3'
3'... Gm5CTG(N) 5CA Gm5C...5' | Not determined |
Ecl136II (EcoICRI) FastDigest Ecl136II |
5'...GAGCT
m5C G...3'
3'...CTCGA Gm5C...5' 5'... m5C GAGCT m5C G...3' 3'... Gm5CTCGA Gm5C...5' | Impaired Blocked |
Eco24I (BanII) |
5'...
m5C GRGCY
m5C G...3'
3'... Gm5CYCGR Gm5C...5' | No effect |
Eco31I (BsaI) FastDigest Eco31I |
5'...GGTCT
m5C G...3'
3'...CCAGA Gm5C...5' 5'... m5C GGTCTC...3' 3'... Gm5CCAGAG...5' | No effect Impaired |
Eco32I (EcoRV) FastDigest Eco32I |
5'...
m5C GATAT
m5C G...3'
3'... Gm5CTATA Gm5C...5' | No effect |
Eco47I (AvaII) FastDigest Eco47I |
5'...GGWC
m5C G...3'
3'...CCWG Gm5C...5' | Blocked |
Eco91I (BstEII) FastDigest Eco91I |
5'...
m5C GGTNAC
m5C G...3'
3'... Gm5CCANTG Gm5C...5' | No effect |
EcoRI FastDigest EcoRI |
5'...
m5C GAATTm5C G...3'
3'... Gm5CTTAA Gm5C...5' | Impaired |
FaqI (BsmFI) FastDigest FaqI |
5'...GGGA
m5C G...3'
3'...CCCT Gm5C...5' 5'... m5C GGGAC...3' 3'... Gm5CCCTG...5' | Blocked Blocked |
HincII (HindII) FastDigest HincII |
5'...GTYRA
m5C G...3'
3'...CARYT Gm5C...5' 5'... m5C GTYRA m5C G...3' 3'... Gm5CARYT Gm5C...5' | Impaired Blocked |
HincII (HindII) FastDigest HincII |
5'...GT
m5C GAC...3'
3'...CA Gm5CTG...5' | Impaired |
HinfI FastDigest HinfI |
5'...GANT
m5C G...3'
3'...CTNA Gm5C...5' 5'... m5C GANT m5C G...3' 3'... Gm5CTNA Gm5C...5' | Impaired Impaired |
HphI |
5'...
m5C GGTGA...3'
3'... Gm5CCACT...5' | No effect |
Hpy8I (MjaIV) FastDigest Hpy8I |
5'...GTNNA
m5C G...3'
3'...CANNT Gm5C...5' 5'... m5C GTNNA m5C G...3' 3'... Gm5CANNT Gm5C...5' | Impaired Not determined |
HpyF10VI (MwoI) FastDigest HpyF10VI |
5'...GC(N)
7G
m5C G...3'
3'...CG(N) 7C Gm5C...5' 5'... m5C GC(N) 7G m5C G...3' 3'... Gm5CG(N) 7C Gm5C...5' | Impaired Blocked |
HpyF10VI (MwoI) FastDigest HpyF10VI |
5'...G
m5C G(N)
6GC...3'
3'...C Gm5C(N) 6CG...5' 5'...G m5C G(N) 5m5C GC...3' 3'...C Gm5C(N) 5 Gm5CG...5' | Impaired Not determined |
KpnI FastDigest KpnI |
5'...
m5C GGTAC
m5C G...3'
3'... Gm5CCATG Gm5C...5' | No effect |
KspAI (HpaI) FastDigest KspAI |
5'...GTTAA
m5C G...3'
3'...CAATT Gm5C...5' 5'... m5C GTTAA m5C G...3' 3'... Gm5CAATT Gm5C...5' | Impaired Blocked |
LguI (SapI) FastDigest LguI |
5'...
m5C GCTCTTC...3'
3'... Gm5CGAGAAG...5' 5'...GCTCTT m5C G...3' 3'...CGAGAA Gm5C...5' | No effect No effect |
Lsp1109I (BbvI) FastDigest Lsp1109I |
5'...
m5C GCAG
m5C G...3'
3'... Gm5CGTC Gm5C...5' | No effect |
LweI (SfaNI) |
5'...GCAT
m5C G...3'
3'...CGTA Gm5C...5' | Impaired |
MbiI (BsrBI) FastDigest MbiI |
5'...
m5C GAGCGG...3'
3'... Gm5CTCGCC...5' | Blocked |
MboI FastDigest MboI |
5'...
m5C GAT
m5C G...3'
3'... Gm5CTA Gm5C...5' | No effect |
MboII FastDigest MboII |
5'...
m5C GAAGA...3'
3'... Gm5CTTCT...5' | No effect |
MnlI FastDigest MnlI |
5'...CCT
m5C G...3'
3'...GGA Gm5C...5' | No effect |
MssI (PmeI) FastDigest MssI |
5'...GTTTAAA
m5C G...3'
3'...CAAATTT Gm5C...5' 5'... m5C GTTTAAA m5C G...3' 3'... Gm5CAAATTT Gm5C...5' | No effect No effect |
Mva1269I (BsmI) FastDigest Mva1269I |
5'...GAATG
m5C G...3'
3'...CTTAC Gm5C...5' 5'... m5C GAATGC...3' 3'... Gm5CTTACG...5' | No effect No effect |
NheI FastDigest NheI |
5'...GCTAG
m5C G...3'
3'...CGATC Gm5C...5' 5'... m5C GCTAG m5C G...3' 3'... Gm5CGATC Gm5C...5' | Impaired Blocked |
NmuCI (Tsp45I) FastDigest NmuCI |
5'...GTCA
m5C G...3'
3'...CAGT Gm5C...5' 5'... m5C GTCA m5C G...3' 3'... Gm5CAGT Gm5C...5' | Impaired Impaired |
OliI (AleI) FastDigest OliI |
5'...CA
m5C GNN
m5C GTG...3'
3'...GT Gm5CNN Gm5CAC...5' 5'...CA m5C GNNNGTG...3' 3'...GT Gm5CNNNCAC...5' | Impaired No effect |
PaeI (SphI) FastDigest PaeI |
5'...
m5C GCATG
m5C G...3'
3'... Gm5CGTAC Gm5C...5' | No effect |
PdmI (XmnI) FastDigest PdmI |
5'...GAA(N)
4TT
m5C G...3'
3'...CTT(N) 4AA Gm5C...5' 5'... m5C GAA(N) 4TT m5C G...3' 3'... Gm5CTT(N) 4AA Gm5C...5' | Impaired Blocked |
PfeI (TfiI) FastDigest PfeI |
5'...GAWT
m5C G...3'
3'...CTWA Gm5C...5' | Blocked |
PfoI FastDigest PfoI |
5'...TC
m5C GGGA...3'
3'...AG Gm5CCCT...5' | Blocked |
FastDigest PspFI | 5'...CCCAGm5C G...3' | Impaired |
PsuI (BstYI) FastDigest PsuI |
5'...
m5C GGATC
m5C G...3'
3'... Gm5CCTAG Gm5C...5' | No effect |
PsyI (Tth111I) FastDigest PsyI |
5'...GA
m5C GNNGT
m5C G...3'
3'...CT Gm5CNNCA Gm5C...5' | No effect |
RsaI FastDigest RsaI |
5'...GTA
m5C G...3'
3'...CAT Gm5C...5' 5'... m5C GTA m5C G...3' 3'... Gm5CAT Gm5C...5' | Impaired Blocked |
RseI (MslI) FastDigest RseI |
5'...CA
m5C GNNNRTG...3'
3'...GT Gm5CNNNYAC...5' | No effect |
SacI FastDigest SacI |
5'...
m5C GAGCT
m5C G...3'
3'... Gm5CTCGA Gm5C...5' | No effect |
SatI (Fnu4HI) FastDigest SatI |
5'...G
m5C GGC...3'
3'...C Gm5CCG...5' 5'...GCNG m5C G...3' 3'...CGNC Gm5C...5' | Blocked Blocked |
SchI (MlyI) FastDigest SchI |
5'...GAGT
m5C G...3'
3'...CTCA Gm5C...5' 5'... m5C GAGTC...3' 3'... Gm5CTCAG...5' | No effect No effect |
SduI (Bsp1286I) |
5'...
m5C GDGCH
m5C G...3'
3'... Gm5CHCGD Gm5C...5' | No effect |
SfiI FastDigest SfiI |
5'...
m5C GGCC(N)
5GGC
m5C G...3'
3'... Gm5CCGG(N) 5CCG Gm5C...5' 5'...GGC m5C G(N) 4GGCC...3' 3'...CCG Gm5C(N) 4CCGG...5' | No effect Impaired |
SfiI FastDigest SfiI |
5'...GGC
m5C G(N)
3m5C GGCC...3'
3'...CCG Gm5C(N) 3 Gm5CCGG...5' | Blocked |
SgeI |
5'...
m5C GNG...3'
3'... Gm5CNC...5' 5'... m5C Gm5 C G...3' 3'... Gm5CGm5C...5' | Cleaves only methylated DNA Cleaves only methylated DNA |
SmoI (SmlI) |
5'...CT
m5C GGT...3'
3'...GA Gm5CCA...5' | No effect |
TaaI (HpyCH4III) FastDigest TaaI |
5'...A
m5C GGT...3'
3'...T Gm5CCA...5' | Impaired |
XapI (ApoI) |
5'...
m5C GAATT
m5C G...3'
3'... Gm5CTTAA Gm5C...5' | No effect |
XceI (NspI) FastDigest XceI |
5'...RCATG
m5C G...3'
3'...YGTAC Gm5C...5' 5'... m5C GCATG m5C G...3' 3'... Gm5CGTAC Gm5C...5' | No effect Not determined |
XmiI (AccI) FastDigest XmiI |
5'...GT
m5C GAC...3'
3'...CA Gm5CTG...5' 5'...GTMKA m5C G...3' 3'...CAKMT Gm5C...5' | Blocked Blocked |
m6A = N6-methyladenine.
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
AatII FastDigest AatII |
5'...TGm6ACGTC(N)
4 TGCT...3'
3'...AC TGCAG(N) 4m6ACGA...5' | No effect |
AluI FastDigest AluI |
5'...TGm6AGCT(N)
5 TGCT...3'
3'...AC TCGA(N) 5m6ACGA...5' | Blocked |
BclI FastDigest BclI |
5'...TGm6A TCA(N)
5 TGCT...3'
3'...AC Tm6AGT(N) 5m6ACGA...5' | Blocked |
BglII FastDigest BglII |
5'...TGm6AGATCT(N)
3 TGCT...3'
3'...AC TCTAGA(N) 3m6ACGA...5' | Impaired |
Bpu10I FastDigest Bpu10I |
5'...CCTGm6AGC(N)
6 TGCT...3'
3'...GGAC TCG(N) 6m6ACGA...5' | No effect |
Bsp143I (Sau3AI) FastDigest Bsp143I |
5'...TGm6ATC(N)
6 TGCT...3'
3'...AC TAG(N) 6m6ACGA...5' | No effect |
EcoRI FastDigest EcoRI |
5'...TGm6AATTC(N)
4 TGCT...3'
3'...AC TTAAG(N) 4m6ACGA...5' | No effect |
HincII (HindII) FastDigest HincII |
5'...GTTGm6AC(N)
7 TGCT...3'
3'...CAAC TG(N) 7m6ACGA...5' | Blocked |
HindIII FastDigest HindIII |
5'...TGm6AAGCTT(N)
3 TGCT...3'
3'...AC TTCGAA(N) 3m6ACGA...5' | Impaired |
HinfI FastDigest HinfI |
5'...TGm6ANTC(N)
5 TGCT...3'
3'...AC TNAG(N) 5m6ACGA...5' | Blocked |
HphI |
5'...GGTGm6A(N)
8 TGCT...3'
3'...CCAC T(N) 8m6ACGA...5' | Blocked |
MboI FastDigest MboI |
5'...TGm6ATC(N)
6 TGCT...3'
3'...AC TAG(N) 6m6ACGA...5' | Blocked |
MboII FastDigest MboII |
5'...TGm6AAGA(N)
5 TGCT...3'
3'...AC TTCT(N) 5m6ACGA...5' | No effect |
MluI FastDigest MluI |
5'...TGm6ACGCGT(N)
3 TGCT...3'
3'...AC TGCGCA(N) 3m6ACGA...5' | No effect |
MnlI FastDigest MnlI |
5'...TGm6AGG(N)
6 TGCT...3'
3'...AC TCC(N) 6m6ACGA...5' | Blocked |
NdeI FastDigest NdeI |
5'...TGm6A(N)
4CATA TGCT...3'
3'...AC T(N) 4GTATm6ACGA...5' | No effect |
OliI (AleI) FastDigest OliI |
5'...TGm6A(N)
4CACG TGCT...3'
3'...AC T(N) 4GTGCm6ACGA...5' | Blocked |
PaeI (SphI) FastDigest PaeI |
5'...TGm6A(N)
5GCA TGCT...3'
3'...AC T(N) 5CGTm6ACGA...5' | Blocked |
PagI (BspHI) |
5'...TCATGm6A(N)
8 TGCT...3'
3'...AGTAC T(N) 8m6ACGA...5' | Impaired |
SacI FastDigest SacI |
5'...TGm6AGCTC(N)
4 TGCT...3'
3'...AC TCGAG(N) 4m6ACGA...5' | No effect |
ScaI FastDigest ScaI |
5'...TGm6AGTACT(N)
3 TGCT...3'
3'...AC TCATGA(N) 3m6ACGA...5' | Blocked |
SspI FastDigest SspI |
5'...TGm6AATATT(N)
3 TGCT...3'
3'...AC TTATAA(N) 3m6ACGA...5' | No effect |
TasI (Tsp509I) FastDigest TTasI |
5'...TGm6AATT(N)
5 TGCT...3'
3'...AC TTAA(N) 5m6ACGA...5' | Blocked |
Conventional/FastDigest | Sequence | Cleavage effect |
---|---|---|
Alw21I (BsiHKAI) FastDigest Alw21I |
5'...Am6AC(N)
6G TGCWC...3'
3'...T TG(N) 6Cm6ACGWG...5' | No effect |
Alw44I FastDigest Alw44I |
5'...Am6AC(N)
6G TGCAC...3'
3'...T TG(N) 6Cm6ACGTG...5' | No effect |
BseSI (Bme1580I) FastDigest BseSI |
5'...Am6AC(N)
6G TGCMC...3'
3'...T TG(N) 6Cm6ACGKG...5' | Impaired |
DraI FastDigest DraI |
5'...TTTAAm6AC(N)
6G TGC...3'
3'...AAATT TG(N) 6Cm6ACG...5' | Blocked |
HincII (HindII) FastDigest HincII |
5'...GTYAm6AC(N)
6G TGC...3'
3'...CART TG(N) 6Cm6ACG...5' | Blocked |
KspAI (HpaI) FastDigest KspAI |
5'...GTTAm6AC(N)
6G TGC...3'
3'...CAAT TG(N) 6Cm6ACG...5' | Blocked |
MluI FastDigest MluI |
5'...Am6ACGCGTNNG TGC...3'
3'...T TGCGCANNCm6ACG...5' | No effect |
MssI (PmeI) FastDigest MssI |
5'...GTTTAAm6AC(N)
6G TGC...3'
3'...CAAATT TG(N) 6Cm6ACG...5' | Blocked |
NsbI (FspI) FastDigest NsbI |
5'...Am6AC(N)
6G TGCGCA...3'
3'...T TG(N) 6Cm6ACGCGT...5' | No effect |
OliI (AleI) FastDigest OliI |
5'...Am6ACAC(N)
4G TGC...3'
3'...T TGTG(N) 4Cm6ACG...5' | Blocked |
RseI (MslI) FastDigest RseI |
5'...GCm6ACNNNNRTG TT...3'
3'...CG TGNNNNYACm6AT...5' | Blocked |
Tru1I (MseI) FastDigest Tru1I |
5'...TTAm6AC(N)
6G TGC...3'
3'...AAT TG(N) 6Cm6ACG...5' | Blocked |