Restriction Site determination of Certain Commercial Restriction Enzyme

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

2. Search and collect a dummy DNA sequence from database. Input the keyword in searcharea by using the name of microbial or specifically accession number.For example, used keyword is accession number of AB453966.1 that belong toMethylococcaceae 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 thescreen page will show as below:

4. Open in new tab, open sites that provide restriction site analysis by particular restrictionenzyme such as Restriction Mapper or NEBcutter, or webcutter.For Restriction Mapper site, it can be accessed in http://www.restrictionmapper.org/ and thescreen 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 itnamed methanol dehydrogenase alpha subunit as an example. Dont forget to check listthe 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 Sitesbutton, and the screen page will show as below:

6. Based on this analysis, it is know that DNA sequence encoding methanol dehydrogenesisalpha 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 blunt-end 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 andmake 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, PI-PspI, 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.