Gene cloning is molecular technique in which gene of interest is copied to produced many identical copies of it.
In this technique gene of interest is fused into a self-replicating genetic material i.e. plasmid.
The insertion of gene of interest is done using self enzymes known as restriction enzymes. The molecule formed after the insertion of gene of interest with the plasmid is called recombinant DNA. which when inserted into a suitable host (bacteria, yeast), self-replicates and generates a large number of identical copies of the particular gene.
The process of introduction of plasmid into the host cell is known as Transformation.
Steps of DNA cloning
Cutting and pasting DNA:
The restriction enzymes are used to cut and join the pieces of DNA. A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cut DNA into two pieces at or near that site. Depending upon the types of restriction enzymes they produce cut ends with short, single-stranded overhangs or If two molecules have matching overhangs, they can base-pair and stick together.
DNA ligase, is another enzyme which is used to join DNA molecule.
In the process of gene cloning
• The restriction enzymes are used to cut the plasmid
• The target gene are mixed with DNA ligase
• The recombinant DNA (Plasmid) thus formed.
Bacterial transformation and selection
After the formation of recombinant DNA it is transferred to the Host i.e. Bateria or Yeast depending on the types of transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.
After the process of transformation, the selection of host cell is required which is done to check the presence of rDNA or not.
A plasmid used as vector for the transformation typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus helps to select the host cell with presence of rDNA. Bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacteria give rise a colony on the plate ensure the presence of transefer of DNA into it.
Not all colonies will necessarily contain the right plasmid. That’s because, during a ligation, DNA fragments don’t always get “pasted” in exactly the way we intend. Instead, we must collect DNA from several colonies and see whether each one contain the right plasmid. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.
After the selection of bacterial colony with the right plasmid, there is process of growing large culture of plasmid-bearing bacteria. Then, we give the bacteria a chemical signal that instructs them to make the target protein.The bacteria serve as miniature “factories,” churning out large amounts of protein.
A selected colony is grown up in a large culture (e.g., a 1-liter flask). The bacterial growth culture induced to express the gene contained in the plasmid, the expressed gene transcribed into mRNA, and the mRNA then translated into protein. The protein encoded by the gene accumulates inside of the bacteria.
After the production of the protein , the bacterial cells can be lysed to release it. The released protein then purififed using biochemical techniques.
Uses of DNA cloning
rDNA formed through cloning techniques are used for many purposes in molecular biology. A applications includes:
• Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.
• Gene therapy. In the case of patients lack the functional form of a specific gene. Gene therapy is used to provide a normal copy of the gene to the cells of a patient’s body. For example, DNA cloning was used to build plasmids which contains a normal version of the gene that was nonfunctional in body.
• Gene analysis. In the gene analysis the gene cloning is used to build artificial, recombinant versions of genes that helps to understand the function of normal genes in an organism.
• Identification of genes responsible for human diseases: A genetic disease found due to defect in a particular gene. In the process of Gene identification the defective gene causing is identified which leads to an indication of the biochemical basis to the disease, enabling therapies to be designed.
• Gene cloning in agriculture:
With help of gene cloning direct changes can be made to the genotype of a plant. In gene addition, gene cloning helps to alter the characteristics of a plant by adding one or more new genes. In gene subtraction, genetic engineering techniques are used to inactivate one or more of the plant’s existing genes. Thus, the genitically modified crops are formed with the desired applications such as the resistance to herbicides, insect and viral resistance etc. the genetically modified food can also produced with the help of gene cloning.
In antisense technology involves the process in which manipulation of gene expression can be done t treat the various diseases. In this technology, the gene to be cloned is ligated into the vector in reverse orientation. After the transcription of cloned gene, the RNA synthsized is the reverse complement of the mRNA from the normal sequence. This antisense RNA is able to prevent synthesis of the product of the gene it is directed against, this is due to the hybridization between antisense and sense copies. Thus cellular ribonucleases enzymes degrade the double-stranded RNA and it block the expression. It prevents ribosomal attachment to sense strand.
The most important application of gene cloning is DNA Sequencing. DNA sequencing includes the determination of the whole nucleotides present within the DNA molecule.