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Ligation of DNA cloning vectors

Update time : 2020-04-15

Source : General Biosystems

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Ligation of DNA cloning vectors

  After obtaining exogenous DNA containing the gene of interest through different channels and selecting or rebuilding an appropriate cloning vector, the next step is how to connect the exogenous DNA with the vector DNA, that is, the in vitro recombination of DNA. This DNA recombination relies on DNA ligase to covalently link the foreign DNA to the carrier. Before reconstructing the vector and starting to connect the foreign gene to the vector, we must combine the research purpose and the characteristics of the gene of interest to carefully design the final recombinant molecule. It should be said that this is a highly technical job, in addition to technical issues, it also involves a profound understanding of the field of recombinant DNA technology. The following is a brief introduction to the connection method.

  Sticky end connection

  The same restriction enzyme cleavage site joins different DNA fragments cleaved by the same restriction endonuclease with identical ends. As long as the enzyme cleaves the DNA and produces single-stranded mutations (5 'overhang and 3' overhang) sticky ends, and the DNA sequence near the cleavage site does not affect ligation, then when these two DNA fragments anneal together, the sticky ends Base pairing between single strands, and then under the catalytic action of DNA ligase to form covalently combined recombinant DNA molecules.

  Different restriction endonuclease sites connect DNA fragments cut by two different restriction endonucleases, and have the same type of sticky ends, that is, compatible ends, and sticky end ligation can also be performed. For example, MboI (GATC) and BamHI (GGATCC) can produce 5 'overhanging GATC sticky ends after cutting DNA, which can be connected to each other.

  Flat end connection

  DNA ligase can catalyze the ligation between blunt ends cleaved by the same and different restriction endonucleases. In principle, the blunt ends produced by restriction enzymes after cutting DNA are also compatible ends and can be connected to each other; if the resulting sticky ends are treated with special enzymes, the single-strand protrusions are filled or flattened to become blunt ends, which can also be implemented Flat end connection.

  Tail link

  Homopolymer tail connection is to use homopolymer sequence, such as annealing between poly A and poly T to complete the connection. Under the action of a terminal transferase, a sticky end is created at the end of the DNA fragment, and then the sticky end is ligated. This is a method of artificially improving the connection efficiency, and also belongs to a special form of sticky end connection.

  Manual joint connection

  For blunt-end DNA fragments or carrier DNA, a phosphorylated linker or appropriate molecule can be ligated to the blunt-end before ligation, so that a new restriction enzyme site is generated. The restriction enzyme that recognizes the new site is then used to excise the distal end of the linker, resulting in a sticky end.