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The following points highlight the top two ways for inserting our gene of interest. The ways are: 1. By the Help of a Ligase Enzyme 2. By Carrying Out the Process of Site Specific Recombination (Cloning with Recombinase System).
Inserting our Gene of Interest: Way # 1.
By the Help of a Ligase Enzyme:
This is the enzymatic process and mostly carried out in RDT experiments. The cohesive ends generated by some RE will anneal (join) themselves by forming hydrogen bonds. But the segments thus annealed are weak and do not withstand experimental conditions. To get a stable joining, the DNA should be joined by using an enzyme called ligase.
DNA ligase joins the DNA molecule covalently by catalysing the formation of phosphodiester bonds between adjacent nucleotides. This is very important to note that sticky ends increases the efficiency of ligation. This is because compatible sticky ends can base pair with one another by hydrogen bonding forming a relatively stable structure for the enzyme to work on.
But what if we have blunt end? It has been found that the efficiency of ligation in case of blunt ends is not good enough to meet the expectations of RDT experiments. Hence, to increase the frequency of ligation in case of blunt end DNA fragments sticky ends are put at their ends.
Putting Sticky Ends onto a Blunt-Ended Molecule:
The sticky ends can be produced by digesting both the vector and the DNA to be cloned with same restriction endonuclease, or with different enzymes that produce the same sticky end. But it is not always possible to do this.
Many times we find vector with sticky ends, but the DNA fragments to be cloned are blunt-ended. Under these circumstances one of the three methods can be used to put the correct sticky ends onto the DNA fragments.
(a) Linkers:
Linkers are short pieces of ds DNA, of known nucleotide sequence, that are synthesized in the test tube. It is blunt- ended but contains a restriction site (BamHI in the example shown in the diagram). DNA ligase can attach linkers to the ends of larger blunt-ended DNA molecules.
Although a blunt end ligation, this particular reaction can be performed very efficiently because synthetic oligonucleotides, such as linkers, can be made in very large amounts and added into the ligation mixture at a high concentration.
More than one linker will attach to each end of the DNA molecule, producing the chain structure. However, digestion with BamHI cleaves the chains at the recognition sequences, producing a large number of cleaved linkers and the original DNA fragment, now carrying BamHI sticky ends. This modified fragment is ready for ligation into a cloning vector restricted with BamHI.
Drawback of linkers:
If the blunt-ended molecule shown in above has more than one restriction sites for BamHI then the restriction step needed to cleave the linkers and to produce the sticky ends would also cleave the blunt-ended molecule.
The resulting fragments will have the correct sticky ends, but that is no consolation if the gene contained in the blunt-ended fragment has now been broken into pieces. The adaptors are used to solve this problem.
(b) Adaptors:
Adaptors, like linkers, are short synthetic oligonucleotides. But unlike linkers, an adaptor is synthesized so that it already has one sticky end. Using adaptors has one more problem. The sticky ends of individual adaptor molecules could base pair with each other to form dimmers [Fig 5.3 (b)].
This left the new DNA molecule still blunt-ended. The sticky ends could be recreated by digestion with a restriction endonuclease, but that would defeat the purpose of using adaptors in the first place.
To solve this problem we remove the phosphate group from the 5′ position of sticky ends of the adaptors by the help of alkaline phosphatase enzyme. Due to this DNA ligase is now unable to form a phosphodiester bridge between 5′-OH and 3′-OH ends.
The result of this manipulation is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized between them by ligation. Adaptors can, therefore, be ligated to a blunt- ended DNA molecule but not to themselves.
After the adaptors have been attached, the abnormal 5′-OH terminus is converted to the natural 5′-P form by treatment with the enzyme polynucleotide kinase, producing a sticky-ended fragment that can be inserted into an appropriate vector.
(c) Homopolymer Tailing:
A homopolymer is simply a polymer in which all the sub-units are the same. Example of poly (dG) homopolymer is made of only de-oxy-guanosine residues. In this technique of ligation of blunt-ended molecule the enzyme terminal deoxynucleotidyl transferase adds a series of similar nucleotides (like only dG or dA) onto the 3′-OH termini of a double-stranded DNA molecule.
In order to ligate together two tailed molecules, the homopolymers must be complementary. Frequently poly-de-oxy-cytosine (poly (dC)) tails are attached to the vector and poly (dG) to our gene of interest (insert DNA). Base pairing between the two occurs when the DNA molecules are mixed.
Inserting our Gene of Interest: Way # 2.
By Carrying Out the Process of Site Specific Recombination (Cloning with Recombinase System):
When we do not have a restriction site for any restriction endonuclease or when the chance mutation for the creation of a restriction site can change the open reading frame (ORF) of our gene of interest, then we follow the recombinase system.
This strategy for the integration of the DNA insert into the vector is also followed when we target at cloning the PCR products or during transferring the DNA insert from one vector into another vector. There are three types of recombinase systems -echo system, creator system and gateway system.