• Effect of Dam Methylation on DNA Cleavage
  
  • Completely Overlapping Dam Methylation
  • Overlapping Dam Methylation
• Effect of Dcm Methylation on DNA Cleavage
  
  • Completely Overlapping Dcm Methylation
  • Partially Overlapping Dcm Methylation
• Effect of CpG Methylation on DNA Cleavage
  
  • Completely Overlapping CpG Methylation
  • Partially Overlapping CpG Methylation
• Effect of EcoKI and EcoBI Methylation on DNA Cleavage
  • EcoBI Overlapping Methylation
    
  • EcoKI Overlapping Methylation

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).
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. E.coli GM2163 dam-, dcm- (#M0099) is available upon request with any product purchase. 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 Fermentas have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA.

Table 1. Fermentas 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

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.
8. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, Nucleic Acids Res., 5, 3231-3236, 1978.
9. Bird, A.P., et al., Methylated and unmethylated DNA compartments in the sea urchin genome, Cell, 17, 889-902, 1979.
10. McClelland, M., The frequency and distribution of methylatable DNA sequences in leguminous plant protein coding genes, J. Mol. Evol., 19, 346-354, 1983.
11. Dreiseikelmann, B., et al., The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis, Biochim. Biophys. Acta, 562, 418-428, 1979.
12. Butkus, V. et al., Investigation of restriction-modification enzymes from M.varians RFL19 with a new type of specificity toward modification of substrate, Nucleic Acids Res., 13, 5727-5746, 1985.

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® Sau3AI (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® AsiSI (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
BdaI	5'...TGm6A TC(N)4TCA...3' 3'...AC Tm6AG(N)4AGT...5'	Blocked
BseJI (BsaBI) FastDigest® BsaBI (BseJI)	5'...Gm6A TC(N)3ATC...3' 3'...C Tm6AG(N)3TAG...5'	Blocked
Bsh1285I (BsiEI) FastDigest® BsiEI (Bsh1285I)	5'...CGm6A TCG...3' 3'...GC Tm6AGC...5'	Impaired
Bsp68I (NruI) FastDigest® NruI (RruI)	5'...Gm6A TCGCGm6A TC...3' 3'...C Tm6AGCGC Tm6AG...5'	No effect
Bsu15I (ClaI) FastDigest® ClaI (Bsu15I)	5'...ATCGm6A TC...3' 3'...TAGC Tm6AG...5'	Blocked
FspBI (BfaI) FastDigest® BfaI (FspBI)	5'...CTAGm6A TC...3' 3'...GATC Tm6AG...5'	No effect
Hin4I	5'...Gm6A TC(N)4VTC...3' 3'...C Tm6AG(N)4BAG...5' 5'...GAY(N)4Gm6A TC...3' 3'...CTR(N)4C Tm6AG...5'	Blocked Blocked
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
TstI	5'...CAC(N)4Gm6A TCC...3' 3'...GTG(N)4C Tm6AGG...5'	Blocked
XbaI FastDigest® XbaI	5'...TCTAGm6A TC...3' 3'...AGATC Tm6AG...5'	Blocked

Table 4. Completely overlapping Dcm (CCWGG) methylation and recognition sites.
Overlapping methylase sequence is highlighted. m5C = 5-methylcytosine.

Conventional/FastDigest®	Sequence	Cleavage effect
FastDigest® SexAI (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® ScrFI (Bme1390I)	5'...Cm5CW GG...3' 3'...G GWm5CC...5'	Blocked
BseDI (BsaJI) FastDigest® BsaJI (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® BslI (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® Bme1580I (BseSI)	5'...GKGCCm5CW GG...3' 3'...CMCGG GWm5CC...5'	No effect
BshNI (BanI) FastDigest® BanI (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® NlaIV (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® HaeIII (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® AlwNI (CaiI)	5'...CAGNNCm5CT GG...3' 3'...GTCNNG GTm5CC...5'	Blocked
Cfr13I (Sau96I) FastDigest® Sau96I (Cfr13I)	5'...GGNCm5CW GG...3' 3'...CCNG GWm5CC...5'	Blocked
CfrI (EaeI)	5'...YGGCm5CW GG...3' 3'...RCCG GWm5CC...5'	Blocked
Eco147I (StuI) FastDigest® StuI (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® AvaII (Eco47I)	5'...GGWCm5CW GG...3' 3'...CCWG GWm5CC...5'	Blocked
Eco57MI	5'...Cm5CT GGAG...3' 3'...G GAm5CCTC...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® BsmFI (FaqI)	5'...Cm5CW GGGAC...3' 3'...G GWm5CCCTG...5'	No effect
GsuI (BpmI) FastDigest® BpmI (GsuI)	5'...CTCm5CA GG...3' 3'...GAG GTm5CC...5'	Blocked
Hin1I (BsaHI) FastDigest® BsaHI (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® MscI (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® PpuMI (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® AscI (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
TsoI	5'...TARCm5CA GG...3' 3'...ATYG GTm5CC...5'	No effect
TstI	5'...CAC(N)6TCm5CW GG...3' 3'...GTG(N)6AG GWm5CC...5'	No effect
Van91I (PflMI) FastDigest® PflMI (Van91I)	5'...Cm5CA GG(N)3TGG...3' 3'...G GTm5CC(N)3ACC...5'	Blocked
XagI (EcoNI) FastDigest® EcoNI (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

Table 6. CpG is located inside the recognition site.

Conventional/FastDigest®	Sequence	Cleavage effect
FastDigest® HaeII (BfoI)	RGCGCY	Blocked
AatII FastDigest® AatII	GACGTC	Blocked
AjiI (BmgBI)	CACGTC	Blocked
BauI (BssSI)	CACGAG	No effect
BcnI (NciI) FastDigest® NciI (BcnI)	CCSGG	Impaired
Bsh1236I (BstUI) FastDigest® Bsh1236I	CGCG	Blocked
Bsh1285I (BsiEI) FastDigest® BsiEI (Bsh1285I)	CGRYCG	Blocked
BshTI (AgeI) FastDigest® AgeI (BshTI)	ACCGGT	Blocked
Bsp119I (BstBI) FastDigest® Bsp119I	TTCGAA	Blocked
Bsp68I (NruI) FastDigest® NruI (RruI)	TCGCGA	Blocked
Bsu15I (ClaI) FastDigest® ClaI (Bsu15I)	ATCGAT	Blocked
Cfr10I (BsrFI) FastDigest® BsrFI (Cfr10I)	RCCGGY	Blocked
Cfr42I (SacII)	CCGCGG	Blocked
Cfr9I (XmaI)	CCCGGG	Impaired
CpoI (RsrII) FastDigest® RsrII (CpoI)	CGGWCCG	Blocked
CseI (HgaI) FastDigest® HgaI (CseI)	GACGC	Blocked
Eco105I (SnaBI) FastDigest® SnaBI (Eco105I)	TACGTA	Blocked
Eco47III (AfeI) FastDigest® AfeI (Eco47III)	AGCGCT	Blocked
Eco52I (EagI) FastDigest® EagI (Eco52I)	CGGCCG	Blocked
Eco72I (PmlI) FastDigest® PmlI (Eco72I)	CACGTG	Blocked
Eco88I (AvaI) FastDigest® AvaI (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® BsaHI (Hin1I)	GRCGYC	Blocked
Hin6I (HinP1I) FastDigest® HinP1I (Hin6I)	GCGC	Blocked
HpaII FastDigest® HpaII	CCGG	Blocked
Kpn2I (BspEI) FastDigest® Kpn2I	TCCGGA	Blocked
MauBI FastDigest® MauBI	CGCGCGCG	Blocked
MbiI (BsrBI) FastDigest® BsrBI (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® FspI (NsbI)	TGCGCA	Blocked
PauI (BssHII) FastDigest® BssHII (PteI)	GCGCGC	Blocked
PdiI (NaeI) FastDigest® NaeI (PdiI)	GCCGGC	Blocked
Pfl23II (BsiWI) FastDigest® BsiWI (Pfl23II)	CGTACG	Blocked
Ppu21I (BsaAI)	YACGTR	Blocked
Psp1406I (AclI) FastDigest® AclI (Psp1406I)	AACGTT	Blocked
PvuI FastDigest® PvuI	CGATCG	Blocked
SalI FastDigest® SalI	GTCGAC	Blocked
SfaAI (AsiSI) FastDigest® AsiSI (SfaAI)	GCGATCGC	Blocked
SgrDI	CGTCGACG	Blocked
SgsI (AscI) FastDigest® AscI (SgsI)	GGCGCGCC	Blocked
SmaI FastDigest® SmaI	CCCGGG	Blocked
SmuI (FauI)	CCCGC	Blocked
SsiI (AciI) FastDigest® AciI (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® SfaNI (BmsI)	5'...GCATm5C G...3' 3'...CGTA Gm5C...5' 5'...m5C GGATG...3' 3'... Gm5CCTAC...5'	Impaired No effect
FastDigest® SanDI (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
AasI (DrdI) FastDigest® DrdI (AasI)	5'...m5C GAC(N)6GTC...3' 3'... Gm5CTG(N)6CAG...5' 5'...m5C GAC(N)6GTm5C G...3' 3'... Gm5CTG(N)6CA Gm5C...5'	Impaired Not determined
AasI (DrdI) FastDigest® DrdI (AasI)	5'...GAm5C G(N)5GTC...3' 3'...CT Gm5C(N)5CAG...5' 5'...GAm5C G(N)4m5C GTC...3' 3'...CT Gm5C(N)4 Gm5CAG...5'	Impaired 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® DraIII (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® ApaLI (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® ScrFI (Bme1390I)	5'...Cm5C GGG...3' 3'...G Gm5CCC...5'	Blocked
BoxI (PshAI) FastDigest® PshAI (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® PshAI (BoxI)	5'...GAC(N)4GTm5C G...3' 3'...CTG(N)4CA Gm5C...5'	No effect
BpiI (BbsI) FastDigest® BbsI (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® BlpI (Bpu1102I)	5'...m5C GCTNAGm5C G...3' 3'... Gm5CGANTC Gm5C...5'	No effect
BseDI (BsaJI) FastDigest® BsaJI (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® BsaBI (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® BslI (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® BsrDI (BseMI)	5'...m5C GCAATG...3' 3'... Gm5CGTTAC...5'	No effect
BseSI (Bme1580I) FastDigest® Bme1580I (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® BanI (BshNI)	5'...GGYRCm5C G...3' 3'...CCRYG Gm5C...5' 5'...m5C GGYRCm5C G...3' 3'... Gm5CCRYG Gm5C...5'	Impaired Blocked
BshNI (BanI) FastDigest® BanI (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® Sau3AI (Bsp143I)	5'...GATm5C G...3' 3'...CTA Gm5C...5'	Blocked
BspLI (NlaIV) FastDigest® NlaIV (BspLI)	5'...GGNNCm5C G...3' 3'...CCNNG Gm5C...5' 5'...m5C GGNNCm5C G...3' 3'... Gm5CCNNG Gm5C...5'	No effect Blocked
BspOI (BmtI) FastDigest® BmtI (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® BstZ17I (Bst1107I)	5'...GTATAm5C G...3' 3'...CATAT Gm5C...5' 5'...m5C GTATAm5C G...3' 3'... Gm5CATAT Gm5C...5'	Impaired Blocked
BsuRI (HaeIII) FastDigest® HaeIII (BsuRI)	5'...m5C GGCm5C G...3' 3'... Gm5CCG Gm5C...5'	No effect
BveI (BspMI) FastDigest® BspMI (BveI)	5'...ACCTGm5C G...3' 3'...TGGAC Gm5C...5'	Impaired
Cfr13I (Sau96I) FastDigest® Sau96I (Cfr13I)	5'...GGNCm5C G...3' 3'...CCNG Gm5C...5'	Blocked
CfrI (EaeI)	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® EarI (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® EcoRV (Eco32I)	5'...m5C GATATm5C G...3' 3'... Gm5CTATA Gm5C...5'	No effect
Eco47I (AvaII) FastDigest® AvaII (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® BsmFI (FaqI)	5'...GGGAm5C G...3' 3'...CCCT Gm5C...5' 5'...m5C GGGAC...3' 3'... Gm5CCCTG...5'	Blocked Blocked
Hin4I	5'...m5C GAY(N)5VTC...3' 3'... Gm5CTR(N)5BAG...5' 5'...GAY(N)5VTm5C G...3' 3'...CTR(N)5BA Gm5C...5'	Impaired Impaired
Hin4I	5'...m5C GAY(N)5VTm5C G...3' 3'... Gm5CTR(N)5BA Gm5C...5'	Not determined
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® HpaI (KspAI)	5'...GTTAAm5C G...3' 3'...CAATT Gm5C...5' 5'...m5C GTTAAm5C G...3' 3'... Gm5CAATT Gm5C...5'	Impaired Blocked
LguI (SapI) FastDigest® SapI (LguI)	5'...m5C GCTCTTC...3' 3'... Gm5CGAGAAG...5' 5'...GCTCTTm5C G...3' 3'...CGAGAA Gm5C...5'	No effect No effect
Lsp1109I (BbvI) FastDigest® BbvI (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® BsrBI (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® AleI (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® SphI (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® TfiI (PfeI)	5'...GAWTm5C G...3' 3'...CTWA Gm5C...5'	Blocked
PfoI FastDigest® PfoI	5'...TCm5C GGGA...3' 3'...AG Gm5CCCT...5'	Blocked
PpiI	5'...m5C GAAC(N)5CTC...3' 3'... Gm5CTTG(N)5GAG...5' 5'...GAAC(N)5CTm5C G...3' 3'...CTTG(N)5GA Gm5C...5'	No effect No effect
PpiI	5'...GAAm5C G(N)4CTC...3' 3'...CTT Gm5C(N)4GAG...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® MslI (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® Fnu4HI (SatI)	5'...Gm5C GGC...3' 3'...C Gm5CCG...5' 5'...GCNGm5C G...3' 3'...CGNC Gm5C...5'	Blocked Blocked
SchI (MlyI) FastDigest® MlyI (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
TstI	5'...CAm5C G(N)5TCC...3' 3'...GT Gm5C(N)5AGG...5' 5'...CAC(N)5TCm5C G...3' 3'...GTG(N)5AG Gm5C...5'	No effect No effect
XapI (ApoI)	5'...m5C GAATTm5C G...3' 3'... Gm5CTTAA Gm5C...5'	No effect
XceI (NspI) FastDigest® NspI (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® AccI (XmiI)	5'...GTm5C GAC...3' 3'...CA Gm5CTG...5' 5'...GTMKAm5C G...3' 3'...CAKMT Gm5C...5'	Blocked Blocked

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® Sau3AI (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® AleI (OliI)	5'...TGm6A(N)4CACG TGCT...3' 3'...AC T(N)4GTGCm6ACGA...5'	Blocked
PaeI (SphI) FastDigest® SphI (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® Tsp509I (TasI)	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® ApaLI (Alw44I)	5'...Am6AC(N)6G TGCAC...3' 3'...T TG(N)6Cm6ACGTG...5'	No effect
BseSI (Bme1580I) FastDigest® Bme1580I (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® HpaI (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® FspI (NsbI)	5'...Am6AC(N)6G TGCGCA...3' 3'...T TG(N)6Cm6ACGCGT...5'	No effect
OliI (AleI) FastDigest® AleI (OliI)	5'...Am6ACAC(N)4G TGC...3' 3'...T TGTG(N)4Cm6ACG...5'	Blocked
RseI (MslI) FastDigest® MslI (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.

Single letter code

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