Restriction Site determination of Certain Commercial Restriction Enzyme

1. Browse to bioinformatics database site via internet such as National Center for
Biotechnological Information (NCBI) or European Nucleotide Archive (ENA) - European
Bioinformatics Institute.
For NCBI GenBank, it can be accessed in http://www.ncbi.nlm.nih.gov/genbank/ and the
screen page will show as below:

2. Search and collect a dummy DNA sequence from database. Input the keyword in search
area by using the name of microbial or specifically accession number.
For example, used keyword is accession number of AB453966.1 that belong to
Methylococcaceae bacterium ET-SHO, and the screen page will showed as below:

3. Open DNA sequence in FASTA format by clicking FASTA buttons in the screen, and the
screen page will show as below:

4. Open in new tab, open sites that provide restriction site analysis by particular restriction
enzyme such as Restriction Mapper or NEBcutter, or webcutter.
For Restriction Mapper site, it can be accessed in http://www.restrictionmapper.org/ and the
screen page will show as below:

5. Copy whole 560 bp of DNA sequence from GenBank and paste it to Sequence Info area.
Naming of the sequence is possible by entering name in Name Your Sequence area, as it
named methanol dehydrogenase alpha subunit as an example. Dont forget to check list
the All Commercial in Include section that mean the site resctriction analysis will simulated

by all listed commercially-available restriction enzyme. After that, press the Map Sites
button, and the screen page will show as below:

6. Based on this analysis, it is know that DNA sequence encoding methanol dehydrogenesis
alpha subunit in Methanococcaceae bacterium ET-SHO may be cut by several enzymes.
Particular restriction enzyme cut the DNA sequence once or several time to forming bluntend or overhang-end sequence at certain recognition sequence, as shown in table below:

While, recognition sequence of several restriction enzymes can not find in sample of DNA and
make them as noncutter i.e. AarI, AbsI, AclI, AflII, AflIII, AgeI, AgsI, AjuI, AlfI, AloI, AlwNI, ApaI,
ApaLI, ApoI, ArsI, AscI, AsuII, AvaI, AvrII, BaeI, BalI, BamHI, BarI, BbvCI, BcgI, BclI, BdaI, BglI,
BglII, BplI, Bpu10I, BsaAI, BsaXI, BsePI, BseRI, BseSI, BseYI, BsgI, BsmI, Bsp1407I, BspMI,
BsrBI, BsrDI, BstXI, BtgZI, BtsI, ClaI, CspCI, DraII, DraIII, DrdI, Eco31I, Eco47III, EcoNI,
EcoP15I, EcoRI, EcoRV, FalI, FseI, FspAI, GsuI, HaeII, HindIII, HpaI, KpnI, MauBI, MfeI, MluI,
NarI, NdeI, NheI, NmeAIII, NotI, OliI, PacI, PasI, PflMI, PfoI, PmaCI, PmeI, PpiI, PpuMI, PsiI, PIPspI, PspXI, PstI, PvuI, SacI, SacII, SanDI, SapI, ScaI, PI-SceI, SexAI, SfiI, SgfI, SgrDI, SmaI,
SnaBI, SpeI, SrfI, Sse8387I, SspI, StuI, SwaI, TaqII, TatI, TfiI, TsoI, TspRI, TstI, VspI, XbaI,
XhoI, XhoII, and XmnI.