Lecture 17 - Alternative cloning strategies PDF

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Isabel Murillo...

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Alternative cloning strategies! Conventional cloning Conventional cloning normally involves the following steps:! 1. Gene of interest amplified by PCR! 2. PCR product and vector digested by REs! 3. Ligation of PCR product to vector ! 4. Transformation ! 5. Plasmic isolation ! 6. Verification by sequencing ! This whole process can take up to a week, this can be time consuming if it needs to be done several times. Having so many steps also increases cost. Alternative cloning strategies need to reduce the cost, reduce the time and increase efficiency of each step. ! Alternative cloning strategies can be split into two main categories:!

- Ligation dependent cloning - includes the conventional cloning method, HiFi, TA cloning, Gibson and Golden Gate assembly !

- Ligation independent cloning - includes sequence and ligation independent cloning (SLIC), Gateway, invivo cloning and in-fusion cloning ! ! Golden gate assembly In golden gate assembly, more than one DNA fragment are assembled into a vector as a single piece using type II endonucleases. The DNA fragments and vector are cut with REs that leave complementary sticky ends so after annealing, no RE sites are left over - there is no scar from cloning. It takes around 3 days to obtain transformed colonies. Golden gate assembly has no buffer incompatibility issues as the same RE is used, and no PCR or Gell purification steps are needed. However, there may be RE sites within the gene of interest, and it is less sequence-independent than overlap-dependent methods of assembly. ! TA cloning In TA cloning, Taq polymerase is used to give the gene of interest adenosine overhangs, and the vector thymine overhangs - these complementary ends will spontaneously anneal. TOPO isomerase is then added to seal the nick, this whole process only takes about 5 minutes. This method is already fast, and TOPO-TA can produce successful ligations in as little as 30 seconds. However this technique is expensive, costing £200+ for 10 reactions, vector choice is limited, and TOPO-TA is not directional, so not all successful inserts will be expressed properly. !

Gibson assembly In seamless cloning methods, exonucleases are used to chew back any insert and vector sequences, so no cloning scars are let over on the recombinant vector - Gibson assembly is a seamless cloning technique. In Gibson assembly, a vector and DNA inserts with overlapping ends are incubated at 50℃ for 15-60 minutes in the Gibson assembly master mix which contains DNA polymerase, DNA ligase and a 5’exonuclease. In the tube, the 5’ exonuclease chews back the 5’ ends, leaving complementary 3’ ends that anneal together. DNA polymerase then extends the 3’ ends to fill in the gaps, and DNA ligase seals the nicks. Gibson assembly is directional, and is very rapid and efficient as many fragments can be combined in a single tube reaction. Also, as Gibson assembly is a seamless cloning method, it won’t affect the open reading frame of the insert. However, Gibson assembly requires primers to be specifically and carefully designed, and is difficult to troubleshoot as it all takes place in one tube. ! Ligation independent cloning (LIC) Ligation independent cloning works with PCR amplified inserts and plasmids that have been made linear by RE digestion or PCR, it uses the 3’-5’ exonuclease activity of T4 DNA polymerase to give the vector and insert complementary overhangs. Firstly, the DNA insert is amplified by PCR using primers with an LIC extension, this PCR product is purified and put into a reaction vessel containing T4 DNA polymerase and one dNTP. T4 DNA polymerase will chew back the insert until it reaches a base corresponding to the dNTP in the reaction vessel, at which point T4’s exonuclease and polymerase activity balance out. Inside a bacterial cells, the vector and insert will ligate, some E. Coli strains are able to repair nicks within their cells. Ligation independent cloning is scarless, low cost, high efficiency, and is able to assemble multiple insert fragments in a single cloning step. However, some types of sequence modifications are not possible e.g adding fusions or tags, and the Gibson method is normally better for single-fragment cloning. ! Infusion Infusion cloning allows ligation-independent cloning of PCR products into any vector in as little as 15 minutes. Infusion has the same principle as LIC, it uses specific primers with 15 base pair extensions homologous to the vector ends, the Infusion enzyme then creates single stranded regions at the ends of the vector and PCR product, which then anneal. Infusion is fast, doesn’t require RE digests or ligations, and is flexible as any vector can be used. However, it can be expensive as custom primers and reaction mixes are required, and occasional mutations in the assembled plasmid can occur. ! ! Hot fusion Hot fusion is a directional and seamless variation of infusion cloning. The vector and PCR product are designed to have complementary 15 base pair extensions. In a single tube, the PCR product, vector and hot fusion master mix are incubated at 50℃ for 1 hour. In this tube, a T5 exonuclease chews back the 5’ ends of the extensions, the complementary 3’ overhangs then anneal, Phusion polymerase extends the 3’ ends and any remaining nicks are sealed by the bacteria....