Question

where do restriction enzymes come from and what is their purpose in nature? List 3 uses of restriction enzymes. Explain how restriction enzymes are involved in the process of cloning a gene

Answer 1

Where do restriction enzyme come from :-

Restriction enzymes are obtained from prokaryotic cells, i.e bacteria as well as archaea.

Their role in nature is to provide immunity to these cells by cleaving the DNA of viruses which infect these cells. So they are component of prokaryotic immune and defense system.

3 uses of Restriction enzyme :-

i) Restriction enzymes are used widely in genetic engineering to clone a DNA fragment into a vector. An enzyme is used to cleave a DNA(gene of interest) by an enzyme whose cleavage sites are present in the flanking region of DNA and then it can be ligated onto a plasmid.

ii) Restriction enzymes are also used to study different alleles by SNPs as difference in two sequences of same Gene from different individuals may means presence of different nucleotides at the same position. This may result in one DNA not getting cleaved by the enzyme while the other will be. This generate a different band pattern on a Gel after electrophoresis and this can be used to study the presence of SNP and their possible role in certain phenotypes.

iii) Restriction enzymes are used in Southern blotting which can be used for DNA fingerprinting. DNA fingerprinting is widely used in forensic science to determine possible victims and culprits of a crime from the DNA containing biological samples obtained from a crime scene. This can be used to determine parentage of children too.

Steps of cloning gene which includes use of Restriction enzymes :-

First to perform a cloning, we have to determine which restriction enzyme can cut our gene of interest from both the sides of it so that it can be separated from the overall DNA.

Once identified, we can use a particular Restriction enzyme to cleave the DNA and remove the gene of interest. Now this gene of interest is first amplified using PCR and then the PCR product can be ligated into a vector.

The vector of our choice usually have multiple cloning sites and these sites contain restriction sites for a wide variety of Restriction enzymes. Now we use the same restriction enzyme as we used to cut our gene out. This is done because mostly those restriction enzymes are used which produce sticky ends. These sticky ends will be complimentary to each other (Gene's sticky end will be complimentary to the vector sticky end) if they are cleaved by the same restriction enzyme. This aids in better ligation as it provide the free ends of DNA to form Hydrogen bond with each other.