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In Biochemistry
Genetic recombination involves the manipulation of genes by using restriction enzymes, where a gene of interest is inserted into a vector to allow for its expression. Genetic recombination is carried out in three steps-1)isolation of the gene of interest(restriction), 2)insertion of gene of interest into the vector to form a recombinant DNA and finally inserting the recombinant DNA into the bacteria. Plasmid is a double stranded extrachromosomal DNA and a vector is a replicon designed to carry with it a gene of interest that would be expressed. The vector consist of an origin of replication, an antibiotic resistance gene(Amp R gene) that permits selection and also has a multiple cloning site where the foreign DNA is inserted. The ampR gene acts as a selection marker used to distinguish between transformants and non-transformants. The bacteria that contains ampR gene will survive ampicillin will survive and those that don’t will die out.

First step of recombination is used to isolate the gene of interest. This is done using restriction endonucleases. The gene of interest is cut using restriction enzymes to produce sticky ends. Restriction enzymes cuts restriction sites of 4-8 bp by hydrolyzing the phosphodiester bonds. This can create both sticky and blunt ends. There are two types of restriction endonucleases-type I and type II.

The final step of recombination is transformation. Several methods are used to form competent cells, which are cells that are able to take the foreign DNA. The process by which scientist determine the

portion of DNA to be cleaved is called restriction mapping. The fragments are separated by gel electrophoresis and determine the site of restriction fragments visualized by staining with ethidium bromide. Ethidium bromide binds to the rungs of the DNA ladder so that when the gel is placed under the UV light, DNA bands can be seen.

After restriction, the gene of interest is inserted into the plasmid using ligation. Ligation uses DNA ligase which uses ATP to drive formation of phosphodiester bond between the 3’OH and 5’phosphate group. The most common ligase used is the T4 ligase from bacteriophage. A bacteriophage requires three ingredients in addition to water. First it needs two more fragments  that have either blunt or sticky ends and a buffer which contains ATP. The buffer is provided or prepared as a 10x concentrate, which after dilution, yields ATP concentration of roughly 0.25mM to 1mM.Ligation reactions are set up and time course ligation reaction is monitored. The reactions are stopped using EDTA at intervals of 3, 10, 20, 30 and 40 seconds.