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Restriction enzymes act as molecular scissors producing cuts at or near sequence specific regions (i.e, restriction sites) at both the strands of a double stranded DNA molecule. On the other hand, DNA ligase enzymes act as molecular glue and help in joining the fragmented DNA pieces together by forming phosphodiester bonds.
What are restriction enzymes?
Restriction enzymes are naturally found in bacterial and archaebacterial cells. Such enzymes protect these unicellular cells by cleaving and destroying foreign genetic particles injected by bacteriophages (i.e, viral molecules responsible for infecting bacterial cells). Classically, there are four types of restriction enzymes i.e, types I, II, III and IV. Example: EcoRI, EcoRII, HindIII, etc.
What is DNA ligase?
DNA ligase, a specific kind of enzyme helps in connecting fragmented DNA strands by facilitating the creation of a phosphodiester bond. It helps the cells to repair single-strand breaks in their duplex DNA, while other varieties (like DNA ligase IV) may only repair double-strand breaks (i.e. a break in both complementary strands of DNA). DNA ligase uses the corresponding strand of the double helix as a template to repair single-strand breaks, producing the last phosphodiester bond necessary to finish the repair of the DNA. Thus, both DNA replication and DNA repair need DNA ligase. Example: T4 DNA ligase, E.coli DNA ligase, etc.
What is molecular cloning?
In this process, a particular gene of interest or the insert can be introduced into a plasmid vector by producing a cut at both the vector and the gene with the same enzyme thus, producing sticky ends and ligating the two fragmented pieces together to produce a recombinant product.
What is the role of the restriction enzymes in molecular cloning?
- The isolated DNA has to be incubated with the specific restriction enzymes under optimal conditions.
- This helps the restriction enzymes in producing sequence specific cuts at specific restriction sites in the DNA molecules. This catalytic reaction is called restriction digestion.
- Agarose Gel Electrophoresis helps in analyzing the progression of the restriction digestion reactions.
- On the other hand, the plasmid vector DNA also has to be processed using similar procedures.
What is the role of the DNA ligase enzymes in molecular cloning?
- Ligation is the process of fusing the two DNA fragments together with the enzyme, DNA ligase.
- The interest molecule and the vector are combined to create the final DNA molecule.
- As a result, the new hybrid DNA molecule is also referred to as a recombinant DNA molecule, and the technique is known as recombinant DNA technology.
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Restriction enzymes are considered as scissors that cleave DNA into fragments at or near specific recognition sites. DNA ligases act as a glue that helps in joining the DNA strands together by catalyzing the formation of a phosphodiester bond.
What are restriction enzymes?
These are protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end. It helps in recombinant DNA technology and genetic engineering.
What are the DNA ligases?
It is a specific type of enzyme that plays a role in, maintaining genomic integrity by repairing single-strand breaks in duplex DNA in living organisms.
In the gene cloning process, Both the plasmid and the DNA sequence of interest are cut with restriction enzymes to generate compatible overhang sticky ends that allow them to bind. The enzyme DNA ligase joins the fragments with matching ends together to form a single, unbroken molecule of DNA. This produces a recombinant plasmid that contains the target gene.
Hence restriction enzymes act as molecular scissors and ligases act as a molecular glue.
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