Molecular cloning refers to the production of the recombinant DNA molecules, this experimental method has developed over the years. This method is achieved by inserting the correct vector gene product and expressing it in the host cell. Molecular cloning provides many advantages, one of these being biopharmaceuticals such as insulin. The cloning of DNA can also be used in the production of human proteins with biomedical applications like insulin, biomedical applications can also apply to others such as recombinant proteins which includes the human growth hormone.
Furthermore, molecular cloning is also known to be used in gene therapy, gene therapy can repair or replace the faulty gene in specific genetic disorders.As mentioned molecular cloning is carried out by the separation of a DNA sequences from a gene and inserted into a vector to undergo proliferation, without changing the original DNA classification. Once the isolation process has been achieved, the molecular clones can be used to produce various amounts of copies of the DNA for the analysis of the gene sequence. This molecular cloning laboratory was illustrated via the following techniques: Restriction digestion of gene PCR product and ligation into a plasmid vector, Transformation of ligations into competent E.
coli cells, PCR screening of transformation of colonies and Gel electrophoresis of PCR samples to identify the recombinant clones. The purpose of these techniques was to separate and purify the plasmid DNA within the cultured cells. The DNA plasmids were determined by treating them with specific reaction endonucleases, this produced a series of DNA fragments. A polymerase chain reaction was accomplished via gel electrophoresis to identify the unknown gene, this had determined the small DNA primers to the specific genes.Gel electrophoresis is a technical process which allows to isolate DNA fragments depending on their characterization of size and charge. This method includes an electrical current running through a gel, the molecules can run through the gel at various speeds and direction this allows them to be detached from each other, however; this depends on their size and charge as mentioned above.
Furthermore, this method includes a ‘loading’ of the DNA into the ‘wells’ in an agrose gel which is submerged in an electrolyte buffer applying electric current. DNA is negatively charge (this is due to the phosphate backbone that it includes) thus it moves away from the anode terminal towards the cathode terminal. This practical was accomplished by visualizing the DNA bands via staining with Nancy 520, this reagent interferes with the double helix thus it fluoresces under UV light.