DNA cloning is the process of making multiple copies of an isolated DNA fragment or fragments using in vitro or in vivo methods. It is used for genetic fingerprinting, genetic engineering, protein production, and genomic sequencing. It is also used in gene therapy to develop new treatments for genetic disorders.
DNA cloning, also known as molecular cloning, gene cloning, and recombinant DNA technology, refers to the process of making multiple copies of an isolated DNA fragment or fragments by in vitro or in vivo methods. You can clone entire gene fragments, random portions of DNA fragments, or specific DNA sequences. In addition to DNA cloning, two other major types of cloning are reproductive cloning, which involves human and animal cloning, and therapeutic cloning, which involves embryonic cloning to harvest stem cells for research purposes and potential medical treatment.
There are various procedures for DNA cloning, but some steps are constant for all. The process begins with the isolation of a DNA fragment or fragments of interest from chromosomal DNA using chemically synthesized restriction enzymes or oligonucleotides. Other methods to accomplish this include several procedures such as polymerase chain reaction (PCR), agarose gel electrophoresis, and DNA sonication.
The isolated DNA fragment now needs to be linked to a primary DNA sequence 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 into it using a ligation procedure, connecting the fragment to a larger piece. The fragments of DNA thus joined artificially are called recombinant DNA.
After the two parts join, the plasmid with the DNA insert is inserted into the host bacterial or mammalian cells. Alternative techniques such as chemical sensitization of cells, electroporation and biolytics can also be used. The plasmid usually contains selectable antibiotic resistance markers and/or color-selective markers, which make it easier to know if cells have been successfully transfected with the DNA insert plasmid. Antibiotic resistance markers only allow cells in which the plasmid has been transfected to grow, and color-selective markers provide visible signs that can be observed.
The transfected cells are cultured and proliferation of the recombinant DNA occurs. 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.
DNA cloning is useful for obtaining information about the genetic composition of an organism and how this affects and influences the vital processes of the organism. DNA cloning is used in genetic fingerprinting; in genetic engineering to create plants with better nutritional value, or better disease resistance, and animals with desirable genetic characteristics; in protein production; and in genomic sequencing to decipher encoded protein or RNA sequences and protein expression.
In gene therapy, DNA cloning is used to develop new treatments for genetic disorders. Recombinant DNA technology has produced over 100 products for human health therapy, such as: insulin for diabetics, factor VIII and factor IX for haemophilia A and B, and erythropoietin (EPO) for the treatment of anemia.
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