Methods of insertion of foreign gene into the vector
There are two types of ligation of foreign gene with plasmid DNA. They are cohesive end ligation and blunt end ligation. The ligation depends upon the nature of the cutting of DNA by the restriction enzyme. The different types of insertion of foreign DNA fragment into the plasmid DNA are explained below:
1) Cohesive end ligation:
The EcoRI cuts the DNA and produces double stranded DNA with cohessive tails. This complementary single stranded tails are often called “Sticky” mortise – and- tanon termini. This property of the enzyme was used to join both ends of a foreign gene with the ends of plasmid DNA.
The Restriction endonucleases like EcoRI, Bam HI, Sau 3A etc cut the DAN around the axis (or) at the line of symmetry of the restriction site. As a result of the cutting, linear double-stranded DNA fragments are formed; each DNA fragment has a single stranded tail at each of both strands. The single stranded ends are ready to form base pairing with each other; hence such DNA fragments are called sticky ended molecules. The same restriction enzyme is also used, to cut the foreign DNA. The complementary bases found at the single-stranded tails of foreign DNA and that those of the vector DNA undergo hydrogen bonding. But hydrogen bonding cannot seal the nick present in between the two DNA fragments. Then an enzyme DNA ligase seals the nick. As a result, the chimeric plasmid DNA is formed. The chimeric plasmid is then inserted into the bacterial cells for bacterial transformation.
2) Blunt end ligation:
Hargobind khorana (1970) first discovered the use of T4-DNA ligase in gene cloning. The blunt end ligation is practiced when Restriction endonucleases cuts the two strands of DNA along the line of symmetry of their restriction sites. Such enzymes produce blunt ended DNA fragments when the DNAs contained in the solution is treated with the restriction enzyme. The DNA fragments do not have sticky ends for ligation. In such cases, both types of DNAs are separately treated with the restriction enzyme, which produces blunt-ended DNA fragments. Then the two types of DNA fragments are mixed together to induce ligation. The ligation reaction is catalyzed by a special group of enzyme known as “T4-DNA ligase”, which joins the blunt-ended molecules. The enzyme links the ends of the foreign DNA fragments. Phosphodiester bond is established between the 3’-hydroxyl group of one fragment and the 5’-phosphate group of another DNA fragment.
Disadvantages:
1) A large number of plasmids are re-circularized by the action of T4-DNA ligase enzyme, the enzyme even links the two ends of the same DNA fragment.
2) The percentage of formation of recombinant plasmids containing the foreign DNA is less than that of homopolymer tailing technique of insertion of foreign gene.
3) Homopolymer tailing:
“P.Lobban” and “Kasier” introduced this method. In this method also the foreign DNA and the plasmid are separately treated with a restriction enzyme to cut DNA into fragments. But this method need not require to produces cohesive ended (or) blunt ended molecules are used to cut the at their restriction site. The enzyme “Terminal nucleotide transferase” is used to add nucleotides to the 3’-hydroxyl group of the DNA fragments. It is a special kind of “polymerase” enzyme, which does not require template strand to add nucleotides to 3’-Hydroxyl group end of the DNA fragments. But the exact sequence of the nucleotides added to the 3,-hydroxyl group depends upon the kind of nucleotides available in the pool.
In this method, both plasmid DNA and foreign DNA are separately treated with a restriction enzyme to produce linear DNA fragments. Then the plasmid DNAs are treated with “Terminal nucleotide transferase” enzyme in presence of ATP molecules. The enzyme adds Adenine nucleotides to the DNA results in the formation of polyadenine tail [Poly (A) tail] at 3’-hydroxyl group end of plasmid DNA fragment.
On the other hand, the foreign DNA fragments are treated with terminal nucleotide transferase in the presence of TMP nucleotides; the enzyme adds thymine nucleotides to 3’-Hydroxyl group of foreign DNA fragments, forms poly thymine tail (poly T) at 3’-hydroxyl end of DNA fragment.
The two kinds of DNA fragments are then mixed together in a solution to establish the insertion of foreign DNA into the plasmid DNA. Complementary base pairing carries this out. The DNA ligase is used to seal the nick found in between the two fragments.
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