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:

  • 5-methylcytosine (m5C)
  • N4-methylcytosine (m4C)
  • N6-methyladenine (m6A)

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).

Effect of Dam Methylation on DNA Cleavage

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:

  • no effect;
  • partial inhibition;
  • complete block.

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.

Table 1. Isoschizomers and Neoschizomers with Differing Sensitivities to Target Methylation.

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

Effect of Dam Methylation on DNA Cleavage

Table 2. Completely overlapping Dam (GATC) methylation and recognition sites.

Overlapping methylase sequence is highlighted. m6A = N6-methyladenine.

Conventional/FastDigest Sequence Cleavage effect
BamHI
FastDigest BamHI
GGm6ATCC
No effect
BclI
FastDigest BclI
TGm6ATCA
Blocked
BglII
FastDigest BglII
AGm6ATCT
No effect
Bsp143I (Sau3AI)
FastDigest Bsp143I
Gm6ATC
No effect
BspPI (AlwI)
GGm6ATC
Blocked
DpnI
FastDigest DpnI
Gm6ATC
Cleaves only Dam methylated DNA
MboI
FastDigest MboI
Gm6ATC
Blocked
PsuI (BstYI)
FastDigest PsuI
RGm6ATCY
No effect
PvuI
FastDigest PvuI
CGm6ATCG
No effect
SfaAI (AsiSI)
FastDigest SfaAI
GCGm6ATCGC
No effect

Table 3. Partially overlapping Dam (GATC) methylation and recognition sites.

Overlapping methylase sequence is highlighted. m6A = N6-methyladenine.

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'...CGm6A TCG...3'
3'...GC Tm6AGC...5'
Impaired
Bsp68I (NruI)
5'...Gm6A TCGCGm6A TC...3'
3'...C Tm6AGCGC Tm6AG...5'
No effect
FastDigest RruI
5'...Gm6A TCGCGm6A TC...3'
3'...C Tm6AGCGC Tm6AG...5'
Impaired
Bsu15I (ClaI)
FastDigest Bsu15I
5'...ATCGm6A TC...3'
3'...TAGC Tm6AG...5'
Blocked
FspBI (BfaI)
FastDigest FspBI
5'...CTAGm6A TC...3'
3'...GATC Tm6AG...5'
No effect
HphI
5'...GGTGm6A TC...3'
3'...CCAC Tm6AG...5'
Blocked
Kpn2I (BspEI)

FastDigest Kpn2I
5'...TCCGGm6A TC...3'
3'...AGGCC Tm6AG...5'

5'...Gm6A TCCGGm6A TC...3'
3'...C Tm6AGGCC Tm6AG...5'
No effect

Not determined
MboII
FastDigest MboII
5'...GAAGm6A TC...3'
3'...CTTC Tm6AG...5'
Blocked
PagI (BspHI)
5'...TCATGm6A TC...3'
3'...AGTAC Tm6AG...5'

5'...Gm6A TCATGm6A TC...3'
3'...C Tm6AGTAC Tm6AG...5'
Impaired

Not determined
PfoI
FastDigest PfoI
5'...TCCNGGm6A TC...3'
3'...AGGNCC Tm6AG...5'
Impaired
SmoI (SmlI)
5'...CTYRAGm6A TC...3'
3'...GARYTC Tm6AG...5'
Impaired
TaqI
FastDigest TaqI
5'...TCGm6A TC...3'
3'...AGC Tm6AG...5'
Blocked
XbaI
FastDigest XbaI
5'...TCTAGm6A TC...3'
3'...AGATC Tm6AG...5'
Blocked

Effect of Dcm Methylation on DNA Cleavage

Table 4. Completely overlapping Dcm (CCWGG) methylation and recognition sites.

Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.

Conventional/FastDigest Sequence Cleavage effect
FastDigest CsiI
ACm5CWGGT
No effect
EcoRII
Cm5CWGG
Blocked
MvaI (BstNI)
FastDigest MvaI
Cm6CWGG
No effect
PasI
CCm5CWGGG
No effect
SgeI
Cm5CWGG
Cleaves only methylated DNA

Table 5. Partially overlapping Dcm (CCWGG) methylation and recognition sites.

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'...GGTACm5CW GG...3'
3'...CCATG GWm5CC...5'

5'...Cm5CW GGTACm5CW GG...3'
3'...G GWm5CCATG GWm5CC...5'
Impaired

Blocked
AloI
5'...GAAC(N)6TCm5CW GG...3'
3'...CTTG(N)6AG GWm5CC...5'
No effect
ApaI
FastDigest ApaI
5'...GGGCCm5CW GG...3'
3'...CCCGG GWm5CC...5'
Impaired
BamHI
FastDigest BamHI
5'...Cm5CW GGATCm5CW GG...3'
3'...G GWm5CCTAG GWm5CC...5'
No effect
BfuI (BciVI)
5'...GTATCm5CW GG...3'
3'...CATAG GWm5CC...5'
No effect
BglI
FastDigest BglI
5'...GCm5CW GGNNGGC...3'
3'...CG 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 GGNCm5CW GG...3'
3'...G GWm5CCNG GWm5CC...5'
Impaired

Blocked
BseSI (Bme1580I)
FastDigest BseSI
5'...GKGCCm5CW GG...3'
3'...CMCGG GWm5CC...5'
No effect
BshNI (BanI)

FastDigest BshNI
5'...GGYRCm5CW GG...3'
3'...CCRYG GWm5CC...5'

5'...Cm5CW GGYRCm5CW GG...3'
3'...G GWm5CCRYG GWm5CC...5'
Impaired

Not determined
Bsp120I (PspOMI)
FastDigest Bsp120I
5'...GGGCCm5CW GG...3'
3'...CCCGG GWm5CC...5'
Blocked
BspLI (NlaIV)

FastDigest BspLI
5'...GGNNCm5CW GG...3'
3'...CCNNG GWm5CC...5'

5'...Cm5CW GGNNCm5CW GG...3'
3'...G GWm5CCNNG 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 GGNNCm5CT GG...3'
3'...G GTm5CCNNG GAm5CC...5'
No effect

Blocked
BsuRI (HaeIII)

FastDigest BsuRI
5'...GGCm5CW GG...3'
3'...CCG GWm5CC...5'

5'...Cm5CW GGCm5CW GG...3'
3'...G GWm5CCG GWm5CC...5'
No effect

No effect
CaiI (AlwNI)
FastDigest CaiI
5'...CAGNNCm5CT GG...3'
3'...GTCNNG GTm5CC...5'
Blocked
Cfr13I (Sau96I)
FastDigest Cfr13I
5'...GGNCm5CW GG...3'
3'...CCNG GWm5CC...5'
Blocked
CfrI (EaeI) 
5'...YGGCm5CW GG...3'
3'...RCCG GWm5CC...5'
Blocked
Eco147I (StuI)
FastDigest Eco147I
5'...AGGCm5CT GG...3'
3'...TCCG GAm5CC...5'
Blocked
Eco24I (BanII)
5'...Cm5CW GGGCCm5CW GG...3'
3'...G GWm5CCCGG GWm5CC...5'
No effect
Eco31I (BsaI)
FastDigest Eco31I
5'...Cm5CW GGTCTC...3'
3'...G GWm5CCAGAG...5'
Impaired
Eco47I (AvaII)
FastDigest Eco47I
5'...GGWCm5CW GG...3'
3'...CCWG GWm5CC...5'
Blocked
Eco91I (BstEII)
FastDigest Eco91I
5'...Cm5CW GGTNACm5CW GG...3'
3'...G GWm5CCANTG GWm5CC...5'
No effect
EcoO109I (DraII)
FastDigest EcoO109I
5'...RGGNCm5CT GG...3'
3'...YCCNG GAm5CC...5'
Blocked
EheI (SfoI)
FastDigest EheI
5'...Cm5CW GGCGCm5CW GG...3'
3'...G GWm5CCGCG GWm5CC...5'
No effect
FaqI (BsmFI)
FastDigest FaqI
5'...Cm5CW GGGAC...3'
3'...G GWm5CCCTG...5'
No effect
GsuI (BpmI)
FastDigest GsuI
5'...CTCm5CA GG...3'
3'...GAG GTm5CC...5'
Blocked
Hin1I (BsaHI)
FastDigest Hin1I
5'...GRCGCm5CW GG...3'
3'...CYGCG GWm5CC...5'
Impaired
HphI
5'...Cm5CW GGTGA...3'
3'...G GWm5CCACT...5'
No effect
KpnI

FastDigest KpnI
5'...GGTACm5CW GG...3'
3'...CCATG GWm5CC...5'

5'...Cm5CW GGTACm5CW GG...3'
3'...G GWm5CCATG GWm5CC...5'
No effect

No effect
MlsI (MscI)
FastDigest MlsI
5'...TGGCm5CA GG...3'
3'...ACCG GTm5CC...5'
Blocked
PfoI
FastDigest PfoI
5'...TCm5CA GGA...3'
3'...AG GTm5CCT...5'
Blocked
Psp5II (PpuMI)
FastDigest Psp5II
5'...RGGWCm5CT GG...3'
3'...YCCWG GAm5CC...5'
Blocked
SduI (Bsp1286I)
5'...Cm5CW GGGCCm5CW GG...3'
3'...G GWm5CCCGG GWm5CC...5'
No effect
SfiI

FastDigest SfiI
5'...GGCm5CW GGNNGGCC...3'
3'...CCG GWm5CCNNCCGG...5'

5'...Cm5CW GGCC(N)5GGCm5CW GG...3'
3'...G GWm5CCGG(N)5CCG GWm5CC...5'
Impaired

No effect
SgsI (AscI)
FastDigest SgsI
5'...Cm5CW GGCGCGCm5CW GG...3'
3'...G GWm5CCGCGCG GWm5CC...5'
No effect
SspDI (KasI)
5'…Cm5CW GGCGCm5CW GG...3'
3'…G GWm5CCGCG 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 GGNCm5CA GG...3'
3'...G GAm5CCNG GTm5CC...5'
No effect

No effect

Effect of CpG Methylation on DNA Cleavage

Table 6. CpG is located inside the recognition site.

Conventional/FastDigest Sequence Cleavage effect
FastDigest BfoI
RGCGCY
Blocked
AatII
FastDigest AatII
GACGTC
Blocked
AjiI (BmgBI)
CACGTC
Blocked
BauI (BssSI)
CACGAG
No effect
BcnI (NciI)
FastDigest BcnI
CCSGG
Impaired
Bsh1236I (BstUI)
FastDigest Bsh1236I
CGCG
Blocked
Bsh1285I (BsiEI)
FastDigest Bsh1285I
CGRYCG
Blocked
BshTI (AgeI)
FastDigest BshTI
ACCGGT
Blocked
Bsp119I (BstBI)
FastDigest Bsp119I
TTCGAA
Blocked
Bsp68I (NruI)
FastDigest RruI
TCGCGA
Blocked
Bsu15I (ClaI)
FastDigest Bsu15I
ATCGAT
Blocked
Cfr10I (BsrFI)
FastDigest Cfr10I
RCCGGY
Blocked
Cfr42I (SacII)
CCGCGG
Blocked
Cfr9I (XmaI)
CCCGGG
Impaired
CpoI (RsrII)
FastDigest CpoI
CGGWCCG
Blocked
CseI (HgaI)
FastDigest CseI
GACGC
Blocked
Eco105I (SnaBI)
FastDigest Eco105I
TACGTA
Blocked
Eco47III (AfeI)
FastDigest Eco47III
AGCGCT
Blocked
Eco52I (EagI)
FastDigest Eco52I
CGGCCG
Blocked
Eco72I (PmlI)
FastDigest Eco72I
CACGTG
Blocked
Eco88I (AvaI)
FastDigest Eco88I
CYCGRG
Impaired
EheI (SfoI)
FastDigest EheI
GGCGCC
Blocked
Esp3I (BsmBI)
CGTCTC
Blocked
FspAI
FastDigest FspAI
RTGCGCAY
Blocked
HhaI
FastDigest HhaI
GCGC
Blocked
Hin1I (BsaHI)
FastDigest Hin1I
GRCGYC
Blocked
Hin6I (HinP1I)
FastDigest Hin6I
GCGC
Blocked
HpaII
FastDigest HpaII
CCGG
Blocked
Kpn2I (BspEI)
FastDigest Kpn2I
TCCGGA
Blocked
MauBI
FastDigest MauBI
CGCGCGCG
Blocked
MbiI (BsrBI)
FastDigest MbiI
GAGCGG
Impaired
MluI
FastDigest MluI
ACGCGT
Blocked
MreI (Sse232I)
FastDigest MreI
CGCCGGCG
Blocked
MspI (HpaII)
FastDigest MspI
CCGG
No effect
NotI
FastDigest NotI
GCGGCCGC
Blocked
NsbI (FspI)
FastDigest NsbI)
TGCGCA
Blocked
PauI (BssHII)
FastDigest PteI
GCGCGC
Blocked
PdiI (NaeI)
FastDigest PdiI
GCCGGC
Blocked
Pfl23II (BsiWI)
FastDigest Pfl23II
CGTACG
Blocked
Ppu21I (BsaAI)
YACGTR
Blocked
Psp1406I (AclI)
FastDigest Psp1406I
AACGTT
Blocked
PvuI
FastDigest PvuI
CGATCG
Blocked
SalI
FastDigest SalI
GTCGAC
Blocked
SfaAI (AsiSI)
FastDigest SfaAI
GCGATCGC
Blocked
SgrDI
CGTCGACG
Blocked
SgsI (AscI)
FastDigest SgsI
GGCGCGCC
Blocked
SmaI
FastDigest SmaI
CCCGGG
Blocked
SsiI (AciI)
FastDigest SsiI
CCGC
Blocked
SspDI (KasI)
GGCGCC
Blocked
TaiI (MaeII)
FastDigest TaiI
ACGT
Blocked
TaqI
FastDigest TaqI
TCGA
No effect
TauI
FastDigest TauI
GCSGC
Blocked
XhoI
FastDigest XhoI
CTCGAG
Impaired

Table 7. CpG partially overlaps the recognition site.

Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.

Conventional/FastDigest Sequence Cleavage effect
FastDigest BmsI
5'...GCATm5C G...3'
3'...CGTA Gm5C...5'

5'...m5C GGATG...3'
3'... Gm5CCTAC...5'
Impaired

No effect
FastDigest KflI
5'...GGGWCCm5C G...3'
3'...CCCWGG Gm5C...5'

5'...CCCAGm5C G...3'
3'...GGGTC Gm5C...5'
Blocked

Impaired
AarI
5'...CACCTGm5C G...3'
3'...GTGGAC Gm5C...5'
Impaired
FastDigest AasI
5'...m5C GAC(N)6GTm5C G...3'
3'... Gm5CTG(N)6CA Gm5C...5'
Not determined
FastDigest AasI
5'...GAm5C G(N)4m5C GTC...3'
3'...CT Gm5C(N)4 Gm5CAG...5'
Not determined
Acc65I (Asp718I)

FastDigest Acc65I
5'...GGTACm5C G...3'
3'...CCATG Gm5C...5'

5'...m5C GGTACm5C G...3'
3'... Gm5CCATG Gm5C...5'
Impaired

Blocked
AdeI (DraIII)

FastDigest AdeI
5'...CAm5C GNNGTG...3'
3'...GT Gm5CNNCAC...5'

5'...CAm5C GNm5C 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)6TGm5C 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)6TCm5C G...3'
3'...CTTG(N)6AG Gm5C...5'
Blocked

No effect
AloI
5'...GAAm5C G(N)5TCC...3'
3'...CTT Gm5C(N)5AGG...5'
No effect
Alw21I (BsiHKAI)
FastDigest Alw21I
5'...m5C GWGCWm5C G...3'
3'... Gm5CWCGW Gm5C...5'
No effect
Alw26I

FastDigest Alw26I
5'...GTCTm5C G...3'
3'...CAGA Gm5C...5'

5'...m5C GTCTC...3'
3'... Gm5CAGAG...5'
No effect

Blocked
Alw44I
FastDigest Alw44I
5'...GTGCAm5C G...3'
3'...CACGT Gm5C...5'
Blocked
ApaI

FastDigest ApaI
5'...GGGCCm5C G...3'
3'...CCCGG Gm5C...5'

5'...m5C GGGCCm5C G...3'
3'... Gm5CCCGG Gm5C...5'
Impaired

Impaired
BamHI
FastDigest BamHI
5'...m5C GGATCm5C G...3'
3'... Gm5CCTAG Gm5C...5'
No effect
BfuI (BciVI)
5'...m5C GTATCC...3'
3'... Gm5CATAGG...5'

5'...GTATCm5C G...3'
3'...CATAG Gm5C...5'
No effect

No effect
BglI

FastDigest BglI
5'...GCm5C G(N)3m5C GGC...3'
3'...CG Gm5C(N)3 Gm5CCG...5'

5'...GCC(N)5GGm5C G...3'
3'...CGG(N)5CC Gm5C...5'
No effect

Impaired
BglI
FastDigest BglI
5'...m5C GCC(N)5GGm5C G...3'
3'... Gm5CGG(N)5CC Gm5C...5'
Blocked
Bme1390I (ScrFI)
FastDigest Bme1390I
5'...Cm5C GGG...3'
3'...G Gm5CCC...5'
Blocked
BoxI (PshAI)

FastDigest BoxI
5'...GAm5C G(N)3GTC...3'
3'...CT Gm5C(N)3CAG...5'

5'...GAm5C GNNm5C GTC...3'
3'...CT Gm5CNN Gm5CAG...5'
Impaired

Blocked
BoxI (PshAI)
FastDigest BoxI
5'...GAC(N)4GTm5C G...3'
3'...CTG(N)4CA Gm5C...5'
No effect
BpiI (BbsI)

FastDigest BpiI
5'...GAAGAm5C G...3'
3'...CTTCT Gm5C...5'

5'...m5C GAAGAC...3'
3'... Gm5CTTCTG...5'
No effect

No effect
BplI
FastDigest BplI
5'...m5C GAG(N)5CTm5C G...3'
3'... Gm5CTC(N)5GA Gm5C...5'
No effect
Bpu10I
FastDigest Bpu10I
5'...CCTNAGm5C G...3'
3'...GGANTC Gm5C...5'
No effect
Bpu1102I (BlpI)
FastDigest Bpu1102I
5'...m5C GCTNAGm5C G...3'
3'... Gm5CGANTC Gm5C...5'
No effect
BseDI (BsaJI)
FastDigest BseDI
5'...Cm5C 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)4ATm5C G...3'
3'...CTA(N)4TA Gm5C...5'

5'...m5C GAT(N)4ATm5C G...3'
3'... Gm5CTA(N)4TA Gm5C...5'
No effect

Not determined
BseLI (BslI)

FastDigest BseLI
5'...Cm5C G(N)6GG...3'
3'...G Gm5C(N)6CC...5'

5'...Cm5C G(N)5m5C GG...3'
3'...G Gm5C(N)5 Gm5CC...5'
Impaired

Blocked
BseMI (BsrDI)
FastDigest BseMI
5'...m5C GCAATG...3'
3'... Gm5CGTTAC...5'
No effect
BseSI (Bme1580I)
FastDigest BseSI
5'...m5C GKGCMm5C G...3'
3'... Gm5CMCGK Gm5C...5'
No effect
BseXI (BbvI)
FastDigest BseXI
5'...m5C GCAGm5C G...3'
3'... Gm5CGTC Gm5C...5'
No effect
BshNI (BanI)

FastDigest BshNI
5'...GGYRCm5C G...3'
3'...CCRYG Gm5C...5'

5'...m5C GGYRCm5C G...3'
3'... Gm5CCRYG Gm5C...5'
Impaired

Blocked
BshNI (BanI)
FastDigest BshNI
5'...GGm5C GCC...3'
3'...CC Gm5CGG...5'
Impaired
Bsp120I (PspOMI)
FastDigest Bsp120I
5'...GGGCCm5C G...3'
3'...CCCGG Gm5C...5'
Blocked
Bsp143I (Sau3AI)
FastDigest Bsp143I
5'...GATm5C G...3'
3'...CTA Gm5C...5'
Blocked
BspLI (NlaIV)

FastDigest BspLI
5'...GGNNCm5C G...3'
3'...CCNNG Gm5C...5'

5'...m5C GGNNCm5C G...3'
3'... Gm5CCNNG Gm5C...5'
No effect

Blocked
BspOI (BmtI)

FastDigest BspOI
5'...GCTAGm5C G...3'
3'...CGATC Gm5C...5'

5'...m5C GCTAGm5C G...3'
3'... Gm5CGATC Gm5C...5'
No effect

No effect
BspPI (AlwI)
5'...GGATm5C G...3'
3'...CCTA Gm5C...5'

5'...m5C GGATC...3'
3'... Gm5CCTAG...5'
No effect

No effect
Bst1107I (BstZ17I)

FastDigest Bst1107I
5'...GTATAm5C G...3'
3'...CATAT Gm5C...5'

5'...m5C GTATAm5C G...3'
3'... Gm5CATAT Gm5C...5'
Impaired

Blocked
BsuRI (HaeIII)
FastDigestBsuRI
5'...m5C GGCm5C G...3'
3'... Gm5CCG Gm5C...5'
No effect
BveI (BspMI)
FastDigest BveI
5'...ACCTGm5C G...3'
3'...TGGAC Gm5C...5'
Impaired
Cfr13I (Sau96I)
FastDigest Cfr13I
5'...GGNCm5C G...3'
3'...CCNG Gm5C...5'
Blocked
CfrI (EaeI)NO SKU
5'...YGGCm5C G...3'
3'...RCCG Gm5C...5'
Blocked
Csp6I (CviQI)
FastDigest Csp6I
5'...m5C GTAm5C G...3'
3'... Gm5CAT Gm5C...5'
No effect
DpnI
FastDigest DpnI
5'...Gm6A Tm5C G...3'
3'...C Tm6A Gm5C...5'
Cleaves only Dam methylated DNA
Eam1104I (EarI)
FastDigest Eam1104I
5'...CTCTTm5C G...3'
3'...GAGAA Gm5C...5'
No effect
Eam1105I (AhdI)

FastDigest Eam1105I
5'...GAm5C G(N)3m5C GTC...3'
3'...CT Gm5C(N)3 Gm5CAG...5'

5'...GAC(N)5GTm5C G...3'
3'...CTG(N)5CA Gm5C...5'
No effect

No effect
Eam1105I (AhdI)
FastDigest Eam1105I
5'...m5C GAC(N)5GTm5C G...3'
3'... Gm5CTG(N)5CA Gm5C...5'
Not determined
Ecl136II (EcoICRI)

FastDigest Ecl136II
5'...GAGCTm5C G...3'
3'...CTCGA Gm5C...5'

5'...m5C GAGCTm5C G...3'
3'... Gm5CTCGA Gm5C...5'
Impaired

Blocked
Eco24I (BanII)
5'...m5C GRGCYm5C G...3'
3'... Gm5CYCGR Gm5C...5'
No effect
Eco31I (BsaI)

FastDigest Eco31I
5'...GGTCTm5C G...3'
3'...CCAGA Gm5C...5'

5'...m5C GGTCTC...3'
3'... Gm5CCAGAG...5'
No effect

Impaired
Eco32I (EcoRV)
FastDigest Eco32I
5'...m5C GATATm5C G...3'
3'... Gm5CTATA Gm5C...5'
No effect
Eco47I (AvaII)
FastDigest Eco47I
5'...GGWCm5C G...3'
3'...CCWG Gm5C...5'
Blocked
Eco91I (BstEII)
FastDigest Eco91I
5'...m5C GGTNACm5C 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'...GGGAm5C G...3'
3'...CCCT Gm5C...5'

5'...m5C GGGAC...3'
3'... Gm5CCCTG...5'
Blocked

Blocked
HincII (HindII)

FastDigest HincII
5'...GTYRAm5C G...3'
3'...CARYT Gm5C...5'

5'...m5C GTYRAm5C G...3'
3'... Gm5CARYT Gm5C...5'
Impaired

Blocked
HincII (HindII)
FastDigest HincII
5'...GTm5C GAC...3'
3'...CA Gm5CTG...5'
Impaired
HinfI

FastDigest HinfI
5'...GANTm5C G...3'
3'...CTNA Gm5C...5'

5'...m5C GANTm5C G...3'
3'... Gm5CTNA Gm5C...5'
Impaired

Impaired
HphI
5'...m5C GGTGA...3'
3'... Gm5CCACT...5'
No effect
Hpy8I (MjaIV)

FastDigest Hpy8I
5'...GTNNAm5C G...3'
3'...CANNT Gm5C...5'

5'...m5C GTNNAm5C G...3'
3'... Gm5CANNT Gm5C...5'
Impaired

Not determined
HpyF10VI (MwoI)

FastDigest HpyF10VI
5'...GC(N)7Gm5C G...3'
3'...CG(N)7C Gm5C...5'

5'...m5C GC(N)7Gm5C G...3'
3'... Gm5CG(N)7C Gm5C...5'
Impaired

Blocked
HpyF10VI (MwoI)

FastDigest HpyF10VI
5'...Gm5C G(N)6GC...3'
3'...C Gm5C(N)6CG...5'

5'...Gm5C G(N)5m5C GC...3'
3'...C Gm5C(N)5 Gm5CG...5'
Impaired

Not determined
KpnI
FastDigest KpnI
5'...m5C GGTACm5C G...3'
3'... Gm5CCATG Gm5C...5'
No effect
KspAI (HpaI)

FastDigest KspAI
5'...GTTAAm5C G...3'
3'...CAATT Gm5C...5'

5'...m5C GTTAAm5C G...3'
3'... Gm5CAATT Gm5C...5'
Impaired

Blocked
LguI (SapI)

FastDigest LguI
5'...m5C GCTCTTC...3'
3'... Gm5CGAGAAG...5'

5'...GCTCTTm5C G...3'
3'...CGAGAA Gm5C...5'
No effect

No effect
Lsp1109I (BbvI)
FastDigest Lsp1109I
5'...m5C GCAGm5C G...3'
3'... Gm5CGTC Gm5C...5'
No effect
LweI (SfaNI)
5'...GCATm5C G...3'
3'...CGTA Gm5C...5'
Impaired
MbiI (BsrBI)
FastDigest MbiI
5'...m5C GAGCGG...3'
3'... Gm5CTCGCC...5'
Blocked
MboI
FastDigest MboI
5'...m5C GATm5C G...3'
3'... Gm5CTA Gm5C...5'
No effect
MboII
FastDigest MboII
5'...m5C GAAGA...3'
3'... Gm5CTTCT...5'
No effect
MnlI
FastDigest MnlI
5'...CCTm5C G...3'
3'...GGA Gm5C...5'
No effect
MssI (PmeI)

FastDigest MssI
5'...GTTTAAAm5C G...3'
3'...CAAATTT Gm5C...5'

5'...m5C GTTTAAAm5C G...3'
3'... Gm5CAAATTT Gm5C...5'
No effect

No effect
Mva1269I (BsmI)

FastDigest Mva1269I
5'...GAATGm5C G...3'
3'...CTTAC Gm5C...5'

5'...m5C GAATGC...3'
3'... Gm5CTTACG...5'
No effect

No effect
NheI

FastDigest NheI
5'...GCTAGm5C G...3'
3'...CGATC Gm5C...5'

5'...m5C GCTAGm5C G...3'
3'... Gm5CGATC Gm5C...5'
Impaired

Blocked
NmuCI (Tsp45I)

FastDigest NmuCI
5'...GTCAm5C G...3'
3'...CAGT Gm5C...5'

5'...m5C GTCAm5C G...3'
3'... Gm5CAGT Gm5C...5'
Impaired

Impaired
OliI (AleI)

FastDigest OliI
5'...CAm5C GNNm5C GTG...3'
3'...GT Gm5CNN Gm5CAC...5'

5'...CAm5C GNNNGTG...3'
3'...GT Gm5CNNNCAC...5'
Impaired

No effect
PaeI (SphI)

FastDigest PaeI
5'...m5C GCATGm5C G...3'
3'... Gm5CGTAC Gm5C...5'
No effect
PdmI (XmnI)

FastDigest PdmI
5'...GAA(N)4TTm5C G...3'
3'...CTT(N)4AA Gm5C...5'

5'...m5C GAA(N)4TTm5C G...3'
3'... Gm5CTT(N)4AA Gm5C...5'
Impaired

Blocked
PfeI (TfiI)
FastDigest PfeI
5'...GAWTm5C G...3'
3'...CTWA Gm5C...5'
Blocked
PfoI
FastDigest PfoI
5'...TCm5C GGGA...3'
3'...AG Gm5CCCT...5'
Blocked
FastDigest PspFI
5'...CCCAGm5C G...3'
3'...GGGTC Gm5C...5'
Impaired
PsuI (BstYI)
FastDigest PsuI
5'...m5C GGATCm5C G...3'
3'... Gm5CCTAG Gm5C...5'
No effect
PsyI (Tth111I)
FastDigest PsyI
5'...GAm5C GNNGTm5C G...3'
3'...CT Gm5CNNCA Gm5C...5'
No effect
RsaI

FastDigest RsaI
5'...GTAm5C G...3'
3'...CAT Gm5C...5'

5'...m5C GTAm5C G...3'
3'... Gm5CAT Gm5C...5'
Impaired

Blocked
RseI (MslI)
FastDigest RseI
5'...CAm5C GNNNRTG...3'
3'...GT Gm5CNNNYAC...5'
No effect
SacI
FastDigest SacI
5'...m5C GAGCTm5C G...3'
3'... Gm5CTCGA Gm5C...5'
No effect
SatI (Fnu4HI)

FastDigest SatI
5'...Gm5C GGC...3'
3'...C Gm5CCG...5'

5'...GCNGm5C G...3'
3'...CGNC Gm5C...5'
Blocked

Blocked
SchI (MlyI)

FastDigest SchI
5'...GAGTm5C G...3'
3'...CTCA Gm5C...5'

5'...m5C GAGTC...3'
3'... Gm5CTCAG...5'
No effect

No effect
SduI (Bsp1286I)
5'...m5C GDGCHm5C G...3'
3'... Gm5CHCGD Gm5C...5'
No effect
SfiI

FastDigest SfiI
5'...m5C GGCC(N)5GGCm5C G...3'
3'... Gm5CCGG(N)5CCG Gm5C...5'

5'...GGCm5C G(N)4GGCC...3'
3'...CCG Gm5C(N)4CCGG...5'
No effect

Impaired
SfiI
FastDigest SfiI
5'...GGCm5C G(N)3m5C GGCC...3'
3'...CCG Gm5C(N)3 Gm5CCGG...5'
Blocked
SgeI
5'...m5C GNG...3'
3'... Gm5CNC...5'

5'...m5C Gm5C G...3'
3'... Gm5C Gm5C...5'
Cleaves only methylated DNA

Cleaves only methylated DNA
SmoI (SmlI)
5'...CTm5C GGT...3'
3'...GA Gm5CCA...5'
No effect
TaaI (HpyCH4III)
FastDigest TaaI
5'...Am5C GGT...3'
3'...T Gm5CCA...5'
Impaired
XapI (ApoI)
5'...m5C GAATTm5C G...3'
3'... Gm5CTTAA Gm5C...5'
No effect
XceI (NspI)

FastDigest XceI
5'...RCATGm5C G...3'
3'...YGTAC Gm5C...5'

5'...m5C GCATGm5C G...3'
3'... Gm5CGTAC Gm5C...5'
No effect

Not determined
XmiI (AccI)

FastDigest XmiI
5'...GTm5C GAC...3'
3'...CA Gm5CTG...5'

5'...GTMKAm5C G...3'
3'...CAKMT Gm5C...5'
Blocked

Blocked

Effect of EcoKI and EcoBI Methylation on DNA Cleavage

Table 8. EcoBI (TGA(N)8TGCT) overlapping methylation.

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

Table 9. EcoKI (AAC(N)6GTGC) overlapping methylation.

m6A = N6-methyladenine.

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 recognition sequences of the following endonucleases may also overlap with DNA sequences methylated by EcoBI and EcoKI.

The following conventional restriction enzymes and the respective FastDigest enzymes have not been tested for sensitivity to EcoBI methylation:

AarI, AdeI, AjiI, Alw21I, BauI, BcuI, BfiI, BglII, BoxI, BpiI, Bpu1102I, BseJI, BseGI, BseMI, BseNI, BseXI, BshTI, Bsp143II, BspPI, Bsu15I, BveI, CaiI, Cfr10I, CseI, DpnI, Eam1104I, Ecl136II, Eco24I, Eco31I, Eco32I, Eco47III, Eco57I, Eco57MI, Eco72I, Eco81I, Eco91I, Eco147I, EcoO109I, Esp3I, FspAI, HindIII, Hin1I, Hin1II, Hin4I, Hpy8I, HpyF3I, LguI, Lsp1109I, LweI, MbiI, Mph1103I, MunI, Mva1269I, NdeI, NmuCI, PdmI, PfeI, Ppu21I, PscI, Psp5II, Psp1406I, PsuI, PsyI, PvuII, RseI, SchI, SduI, SmiI, SmoI, SspI, TaaI, TaiI, TatI, TscAI, TstI, VspI, XagI, XapI and XceI.

The following conventional restriction enzymes and the respective FastDigest enzymes have not been tested for sensitivity to EcoKI methylation:

AanI, AdeI, AjiI, BauI, BcuI, BfiI, BseNI, BshNI, BshTI, Bsp119I, BveI, Cfr10I, Eco72I, Eco91I, FspAI, Hpy8I, PdmI, Ppu21I, PscI, Psp1406I, SduI, TaaI, TaiI, TscAI, TsoI and XceI.

References:

  1. McClelland, M., The effect of sequence specific DNA methylation on restriction endonuclease cleavage, Nucleic Acids Res., 9, 5859-5866, 1981.
  2. McClelland, M., et al., Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases, Nucleic Acids Res., 22, 3640-3659, 1994.
  3. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12, J. Bacteriol., 114, 1143-1150, 1973.
  4. May, M.S., Hattman, S., Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes, J. Bacteriol., 123, 768-770, 1975.
  5. Hattman, S., et al., Sequence specificity of the P1 modification methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene, J. Mol. Biol.,126, 367-380, 1978.
  6. Geier, G.E., Modrich, P., Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease, J. Biol. Chem., 254, 1408-1413, 1979.
  7. Buryanov, Ya.I., et al., Site specificity and chromatographic properties of E.coli K12 and EcoRII DNA-cytosine methylases, FEBS Letters, 88, 251-254, 1978.
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