Question

explain the role of that DNA ligase, restriction enzymes and plasmids have in creating recombination DNA

Answer 1

The plasmids are vectors in which any gene can be cloned as it possesses multiple cloning site, different restriction enzyme sites which can be used to insert a gene, a bacterial origin of replication to replicate and increase the copy number of a cloned gene and antibiotic resistance gene that help only intact plasmids to grow on media that have the antibiotic and thus select for plasmids with cloned inserts. It might also have a mammalian origin of replication for propagation in mammalian cells. It can also have ribosome entry site and other mechanisms for translation so that they can be transcribed and translated to obtain proteins from recombinant cloned genes.

To elucidate the roles of DNA ligase, restriction enzyme, and vector, the process of cloning a gene is described.

• To clone a gene from a human cell, initially, RNA is isolated from the cell, and cDNA is made and if it is a bacterial gene then there is no need for preparation of RNA or cDNA. It can be directly amplified by PCR by using primers.

• Previously designed primers against the gene are used to amplify the gene via PCR. Primers have restriction sites compatible with the vector.

• The amplified product is cut with two different restriction endonucleases to ensure directional cloning.

• The vector in which the gene is to be inserted is cut with the same endonucleases.

• Once both the vector and the PCR product are cut, standard ligation reaction are set up to ensure ligation of a PCR product into the vector which is mediated by the DNA ligase enzyme and a very crucial step of the entire cloning method. unligated vectors do not give colonies while wrongly ligated vectors without the gene of interest give false positive colonies.

• This ligated DNA is now transformed into bacterial cell and the bacterial cells were selected based on antibiotic resistance present in the vector. The bacteria which will grow in the presence of antibiotic will have the gene cloned in vector.