Molecular cloning is the process of inserting a DNA sequence (generally a gene) from a selected species into a replicating vehicles like vectors or plasmids, for propagation, without any alteration or modification of the original DNA sequence.
Molecular clones can be used to express the resulting proteins for the utilization of protein’s functions. It can help us learn more about the function of a particular fragment of DNA or a gene.
Molecular cloning basically involves these steps:
1) Isolation of target DNA fragments or “inserts”.
2) Ligation of the inserts into a plasmid.
3) Transformation of the recombinant plasmid into a host for e.g. Bacteria.
4) Screening of the hosts containing the recombinant plasmid.
Requirements for cloning:
- The first thing that is required for cloning is an insert or a DNA fragment that is intended to clone. It can be extracted from an organism or fossil or can be created artificially.
- The second thing that we need is the vehicle to carry our gene of interest or a vector. A vector is simply a plasmid DNA used as a tool in molecular biology to make multiple copies of our gene of interest or to produce protein from a particular gene. Plasmids are extra chromosomal self-replicating DNA molecules that are present inside of the bacterial cells. A plasmid contains multiple cloning sites that contains recognition sites for restriction endonucleases or the restriction enzymes. Plasmids also contains origin of replication that allows them to replicate independently.
- The third thing that’s needed for cloning is the host for the vector for its mass replication.
- Replicated vectors are then purified from the host bacteria and analysed on a gel.
The Cloning Methodology:
- First, the DNA fragment or the insert for molecular cloning is selected and isolated and then amplified for getting a desired amount of the DNA fragment. The amplification can be done by PCR. The insert can be isolated from any type of cell.
- Once the insert is amplified, insert and vector are then digested with a restriction enzyme or restriction endonucleases.
- After digestion of the insert and the vector they are purified by using gel electrophoresis to purify the vector from uncut or supercoiled vector DNA or small pieces of DNA molecules that were digested by restriction enzymes. This step is called gel purification. Purification of the digested insert is should be carried out by agarose gel electrophoresis when the insert is PCR amplified with the same selective marker. Or otherwise a purification kit can be used.
- After the purification step the insert is ligated to the vector using an enzyme called DNA ligases that can be E.coli DNA Ligase or the bacteriophage T4 DNA Ligase. Ligation involves the formation of phosphodiester bonds between adjacent 5’-phosphate and 3’-hydroxyl residues.
The efficiency of ligation reaction depends on various factors like:
- The concentration of the insert, it should be high enough to ensure intermolecular ligation and not the self-ligation but not too high to promote oligomeric molecule formation.
- The vector insert ratio: the ratio shouldn’t be varying considerably or otherwise it will lower the ligation efficiency. Usually a molar ratio of insert to vector that is used is 1:1, 2:1 and 3:1.
After ligation, transformation is done. Transformation is simply the addition of naked foreign DNA into host bacterium. During this process the bacterial cell will take up the ligated vector and the vector inside of the host body will replicate multiple times as it is capable of self-replication. Finally, the growth of the transformed bacteria will amplify the clones i.e. more copies of the plasmid are formed. A collection of different cloned DNA is called a library.
Plasmids can then be purified and digested with restriction enzyme to confirm the presence of the insert, which will verify the transformation of the plasmid with the insert and not with the self-ligated plasmid. Sequencing can also be done to confirm the cloning of the insert or the gene of insert.
Applications of gene cloning
- Production of recombinant proteins: production of protein from a cloned gene in a defined and non-pathogenic microorganism has so many advantages. Various proteins like insulin that are produced in small amounts in our body can be missing or defective in case of any disease, so these proteins can be produced by cloning of required genes which will then be expressed to produce the protein by the bacterial cells.
- Synthesis of recombinant vaccines: the growth factors are produced in our body in very small amounts so recombinant gene cloning technology can be used to produce these factors and other rare proteins in larger amounts for clinical uses.
- Live recombinant vaccines: recombinant vaccinia viruses can be used as live vaccines against diseases. If a gene coding for a virus coat protein is ligated into the vaccinia genome, in the presence of a vaccinia promoter, then the gene will be expressed. After injection into the blood stream, replication of the recombinant virus will result in production of new vaccinia particles, and in significant number of the major surface antigen.
- Gene therapy: it is the process of insertion, alteration and removal of a segment of DNA from an individual’s DNA to treat a disease. Cloned genes can be introduced into stem cells from the bone marrow.
- Germline therapy: the fertilized egg is ingested with the cloned gene and then it is re-implanted into the mother.