b'Type IIs restriction enzymesA specific group of restriction enzymes called Type IIs endonucleases cleave One eect of this oset cleavage is that the still-intactDNAu onuletssisd teh oefttwhoei rf rraegcmogennittsio hna sveeq bueeennc einst.e Innt icoonmallbyi ndaetsioignn Owed nitheDeNeAc t of this oset cleavage is that the still-intactunless the two fragments have been intentionally designed recognition sequence remains with one fragment while the ligasteo,bTyep ceo ImIs preastitbrliec.t ioWni tehn szuycmhe usp afrroen utt dilizeesdig nto ( ddorinveeby the insreecrtoiognn iotiof on nsee qour ence remains with one fragment while theto be compatible. With such upfront design (done by other fragment loses the recognition sequence. A secondsevecrahlo DoNsiAng f roarg mcreenattsin ign ttoy pae r eIIscipcieonmt pvaetcibtolerwcliothnoinugttvheec tioncrs oluthsieorn f roafg rmeseidntu alol ses the recognition sequence. A secondchoosing or creating type IIscompatible cloning vectors eect is that while the precise distance of the cleavage site restraicntdio ng eennezryamtineg s icteosm apnadti bolteh ecrlo unninwga fnrtaegdm DeNntAssveiaq ugeennce ese aet cfrt aisg mtheant twhile the precise distance of the cleavage siteand generating compatible cloning fragments via gene from the recognition site is enzyme-dependent, as is thejunctsioynntsh (essciasr olers Ps CcRlo npinrimg).er design), workows can befrom the recognition site is enzyme-dependent, as is thesynthesis or PCR primer design), workows can be type of cleavage (blunt, 3 protruding, or 5 protruding), thesimplied and cloning products can be scarless, which type of cleavage (blunt, 3 protruding, or 5 protruding), thesimplied and cloning products can be scarless, which sequence at the cleavage site is typically irrelevant; onceFind cFaans tbDeig aens tim Typpoer tIaIsn te cnoznymsidees raatt ion when dealing withsequence at the cleavage site is typically irrelevant; oncecan be an important consideration when dealing with the enzyme binds to its recognition site, it will cleave anythermopoefins rheeard.cinogm f/rafamsetds.i gIte isst atylspoe piisossible to simultaneouslty he enzyme binds to its recognition site, it will cleave anyopen reading frames. It is also possible to simultaneously DNA sequence the appropriate distance away. The resultassemble multiple inserts in a specic order into aDNA sequence the appropriate distance away. The resultassemble multiple inserts in a specic order into a of this mechanism is that two dierent DNA fragmentsFor GsienngeleA vrte Tcytopre [ 4IIs]. Assembly Kits, go toof this mechanism is that two dierent DNA fragmentssingle vector [4].cleaved by a given type IIs restriction enzyme that leavesthermofisher.com/typeiis cleaved by a given type IIs restriction enzyme that leaves protruding ends will not have compatible overhangs protruding ends will not have compatible overhangsA B A BType II enzyme Type IIs enzyme Type II enzyme Type IIs enzymeCloningN N N N N G A A T T C N N N N N N N N N G A A G A C N N N N N N N N N N N N N N N N N N N G A A T T C N N N N N N N N N G A A G A C N N N N N N N N N N N N N NN N N N N C T T A A G N N N N N N N N N C T T C T G N N N N N N N N N N N N N N N N N N N C T T A A G N N N N N N N N N C T T C T G N N N N N N N N N N N N N NRecognition site and cleavage site of Type IIs versus Type II restriction enzymes.Figure 1. Type II vs. type IIs restriction endonucleases. Representative (A) type II (EcoRI) and (B) type IIs (BpiI) enzymes are shown. The DNA bindFinigg ure 1. Type II vs. type IIs restriction endonucleases. Representative (A) type II (EcoRI) and (B) type IIs (BpiI) enzymes are shown. The DNA binding and DNA cleavage activities of typical type II enzymes are closely tied together in a single protein domain, as is reected in their cleavage patterns. Typae nd IIs DNA cleavage activities of typical type II enzymes are closely tied together in a single protein domain, as is reected in their cleavage patterns. Type IIs enzymes bind at one DNA sequence, but a second protein domain cleaves DNA at a specied distance in a sequence-independent manner. 41enzymes bind at one DNA sequence, but a second protein domain cleaves DNA at a specied distance in a sequence-independent manner.To further illustrate the logic behind Golden Gate cloningThe key is that during repeated cycles between theTo further illustrate the logic behind Golden Gate cloningThe key is that during repeated cycles between the for the transfer of a single DNA fragment (which could be atemperatures optimal for digestion (37C) and ligationfor the transfer of a single DNA fragment (which could be atemperatures optimal for digestion (37C) and ligation PCR product, an Invitrogen TOPOcloned PCR product,(23C), the desired nal product loses its type IIsPCR product, an Invitrogen TOPOcloned PCR product,(23C), the desired nal product loses its type IIs or a synthetic DNA fragment) into a recipient vector,recognition sites when it is generated, while undesiredor a synthetic DNA fragment) into a recipient vector,recognition sites when it is generated, while undesired cloning products or uncut vector and insert fragmentscloning products or uncut vector and insert fragments ends such that cleavage leaves the fragment with tworetain the recognition sites and are subject to digestion ends such that cleavage leaves the fragment with tworetain the recognition sites and are subject to digestion dierent sticky ends, each compatible with the recipientin subsequent cycles. During ligation cycles, potentialdierent sticky ends, each compatible with the recipientin subsequent cycles. During ligation cycles, potential vector, but removes the enzyme-binding sites (Figure 2A).nonproductive outcomes include the possibility that anvector, but removes the enzyme-binding sites (Figure 2A).nonproductive outcomes include the possibility that an The recipient vector must be similarly designedthe twoexcised vector fragment is religated into the same vectoTr hit e recipient vector must be similarly designedthe twoexcised vector fragment is religated into the same vector it enzyme-binding sites are oriented such that the osetwas liberated from or the recipient vector is cleaved at oennly zyme-binding sites are oriented such that the osetwas liberated from or the recipient vector is cleaved at only cleavage of the type IIs enzymes removes the sites fromone of its two type IIs sites and is religated. In these cascelse, avage of the type IIs enzymes removes the sites fromone of its two type IIs sites and is religated. In these cases, the now-linearized vector, and the two sticky ends of thethe enzyme-binding sites would once again be present the now-linearized vector, and the two sticky ends of thethe enzyme-binding sites would once again be present vector are compatible with the directional cloning of thein the starting material and these molecules would bevector are compatible with the directional cloning of thein the starting material and these molecules would be insert, but not with religation of the vector itself. available for cleavage in the next digestion cycle. Howevienrs, ert, but not with religation of the vector itself. available for cleavage in the next digestion cycle. However, if the digested insert is ligated to the recipient vector asif the digested insert is ligated to the recipient vector as desired, the recombinant molecule does not contain thedesired, the recombinant molecule does not contain the type IIs binding sites and the reaction is irreversible. Thetype IIs binding sites and the reaction is irreversible. The'