DNA cloning – also known as molecular cloning, gene cloning and recombinant DNA technology - refers to the process of creating multiple copies of an isolated DNA fragment or fragments by in vitro or in vivo methods. It is possible to clone entire gene fragments, random portions of DNA fragments or specific DNA sequences. Apart from DNA cloning, two other main cloning types are reproductive cloning, which is concerned with human and animal cloning, and therapeutic cloning, concerned with embryonic cloning to harvest stem cells for research and potential medical treatment purposes.
There are various procedures for DNA cloning, but some steps are constant for all. The process begins with isolating a DNA fragment or fragments of interest from the chromosomal DNA using restriction enzymes or chemically synthesized oligonucleotides. Other methods for accomplishing this include different procedures like polymerase chain reaction (PCR), agarose gel electrophoresis and DNA sonication.
The isolated DNA fragment now must be linked to a primary DNA sequence that is capable of replicating and propagating both itself and the DNA fragment linked to it. A restriction enzyme cuts a self-replicating DNA molecule and the isolated DNA fragment is inserted in it by a ligation procedure, connecting the fragment to a larger piece. The DNA fragments thus artificially joined are called recombinant DNA.
After the two parts are joined, the plasmid with the DNA insert is inserted into host bacterial or mammalian cells. Alternative techniques like chemical sensitivation of cells, electroporation and biolistics can also be used. The plasmid usually contains selectable antibiotic resistance markers and/or color selection markers, which make it easier to know if the cells have been successfully transfected with the DNA insert plasmid. The antibiotic resistance markers allow only cells in which the plasmid has been transfected to grow, and the color selection markers provide visible marks that can be observed.
The transfected cells are cultured and proliferation of the recombinant DNA takes place. The resulting clones are genetically identical organisms containing the recombinant DNA. This can be confirmed using PCR, restriction fragment analysis, or other DNA sequencing methods.
Cloning DNA is helpful in getting an insight into an organism's genetic make-up and how this affects and influences the organism's life processes. DNA cloning is being used in genetic finger-printing; in genetic engineering to create plants with better nutritional value, or better resistance to diseases and animals with desirable genetic features; in protein production; and in sequencing genomes to decipher encoded protein or RNA sequences and protein expression.
In gene therapy, DNA cloning is being used to develop new treatments for genetic-related disorders. Recombinant DNA technology has produced over 100 products for human health therapy, such as: insulin for diabetics, factor VIII and factor IX for hemophilia A and B, and erythropoietin (EPO) for treating anemia.
