Following figure shows the restriction enzyme Eco RI, extracted from the bacterium Escherichia coli, attached to a DNA molecule
(i) Outline how restriction enzymes may be used in the formation of recombinant DNA.
(ii) Suggest an advantage to bacteria in having restriction enzymes in the cytoplasm of their cells.
(iii) Name two other enzymes used in the formation of recombinant DNA and briefly describe their role.
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(i) Restriction Enzymes (Molecular scissors) These are enzymes which are used for cutting of DNA at specific locations during rDNA technology.
Restriction enzymes belong to a larger class of enzymes called nucleases, which are of two types:
(a) Exonucleases remove nucleotides from the ends of the DNA (either 5’ or 3’) in one strand of duplex.
(b) Endonucleases make cuts at specific position within DNA.
The first restriction endonuclease named Hind II was isolated by Smith Wilcox and Kelley in 1968 . It was found that it always cuts DNA molecules at a particular point recognising a specific sequence of six base pairs known as the recognition sequence for Hind II.
More than 900 restriction enzymes are known today that have been isolated from over 230 bacterial strains, and each of which recognises different recognition sequences. Restriction , endonucleases are not present in eukaryotic cells.
(ii) The advantage is that the restriction enzymes would not affect the DNA inside the cells themselves if the enzymes were in the cytoplasm of the cells.
(iii) (a) DNA Ligases (Molecular glue) These enzymes repair broken DNA by joining two nucleotides. It is used in genetic engineering to reverse the action of restriction enzymes, i.e. to join complementary DNA strands/ends together, e.g T4 DNA ligase.
(b) Alkaline Phosphatase (AP) This enzyme removes the phosphate group from the 5’ end of a DNA molecule, leaving a free 5’ hydroxyl group, which prevents unwanted self-ligation of vector DNA molecules during the formation of recombinant DNA.