Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vitro. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA. Cloning is used in a wide array of biological experiments and technological applications such as large scale protein production. Overview In essence, in order to amplify any DNA sequence in vivo and in vitro, the sequence in question must be linked to primary sequence elements capable of directing the replication and propagation of themselves and the linked sequence in the desired target host. The required sequence elements differ according to host, but invariably include an origin of replication, and a selectable marker. In practice, however, a number of other features are desired and a variety of specialized cloning vectors exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations that are useful in downstream applications. Recombinase-based cloning A novel procedure of cloning or subcloning of any DNA fragment by inserting the special DNA fragment of interest into a special area of target DNA through interchange of the relevant DNA fragments.[1] This is a one-step reaction: simple, efficient, facilitating high throughput or automatic cloning and/or subcloning.[2] One of the currently popular recombinase-based systems is marketed under the name Gateway Technology Restriction/ligation cloning In the classical restriction and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: DNA fragmentation with restriction endonucleases, ligation of DNA fragments to a vector, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected at various points depending on the particular application; these are summarized as a ‘cloning strategy’. Isolation of insert Initially, the DNA fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be accomplished in a number of alternative ways. Insert preparation is frequently achieved by means of polymerase chain reaction, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis. Chemically synthesized oligonucleotides can also be used if the target sequence size does not exceed the limit of chemical synthesis. Isolation of insert can be done by using shotgun cloning, c-DNA clones, gene machines (artificial chemical synthesis). Transformation Following ligation, the ligation product (plasmid) is transformed into bacteria for propagation. The bacteria is then plated on selective agar to select for bacteria that have the plasmid of interest. Individual colonies are picked and tested for the wanted insert. Maxiprep can be done to obtain large quantity of the plasmid containing the inserted gene. Transfection Following ligation, a portion of the ligation reaction, including vector with insert in the desired orientation is transfected into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation and biolistics. Chemical sensitization of cells is frequently employed since this does not require specialized equipment and provides relatively high transformation efficiencies. Electroporation is used when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly utilized in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells. The bacterial transformation is generally observed by blue white screening. Selection Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that contain the desired insert at the appropriate orientation and isolate these from those not successfully transformed. Modern cloning vectors include selectable markers (most frequently antibiotic resistance markers) that allow only cells in which the vector, but not necessarily the insert, has been transfected to grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (via α-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing. Genetic engineering (for full page see Genetic Engineering) Genetic engineering is a method of changing the inherited characteristics of an organism in a predetermined way by altering its genetic material. This is often done to enable micro-organisms, such as bacteria or viruses, to synthesize increased yields of compounds, to form entirely new compounds, or to adapt to different environments. Other uses of this technology, which is also called recombinant DNA technology, include gene therapy, which is the supply of a functional gene to a person with a genetic disorder or with other diseases such as acquired immune deficiency syndrome (AIDS) or cancer, and the cloning of whole organisms. Genetic engineering involves the manipulation of deoxyribonucleic acid, or DNA. Important tools in this process are restriction endonucleases (so-called restriction enzymes) that are produced by various species of bacteria. Restriction enzymes can recognize a particular sequence of the chain of chemical units, called nucleotide bases, which make up the DNA molecule and cut the DNA at that location. Fragments of DNA generated in this way can be joined using other enzymes called ligases. Restriction enzymes and ligases therefore allow the specific cutting and reassembling of portions of DNA. Also important in the manipulation of DNA are so-called vectors, which are pieces of DNA that can self-replicate (produce copies of themselves) independently of the DNA in the host cell in which they are grown. Examples of vectors include plasmids, viruses, and artificial chromosomes. Vectors permit the generation of multiple copies of a particular piece of DNA, making this a useful method for generating sufficient quantities of material with which to work. The process of engineering a DNA fragment into a vector is called “molecular cloning”, because multiple copies of an identical molecule of DNA are produced. Another way of producing many identical copies of a particular (often short, for example, 100-3,000 base pairs) DNA fragment is the polymerase chain reaction. This method is rapid and avoids the need for cloning DNA into a vector. Gene therapy Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders. The history of human gene therapy is, however, not a particularly happy one. The effect of introducing a gene into cells rarely promotes more than small transient relief from the symptoms of the disease being treated. Worse still, there have been highly publicized cases where gene therapy trial patients have suffered as a consequence of the treatment itself. For example, in 1999 an 18-year-old gene therapy trial volunteer from Philadelphia died following a gene therapy trial. In addition, one of the few success stories of human gene therapy—the treatment of severe combined immune deficiency, X-SCID—has turned out to have unforeseen consequences. Bone marrow cells were taken from patients suffering from this disease and treated with a virus to introduce a functional copy of the defective gene. When the modified bone marrow cells were returned to patients, their immune systems were functional once more. However, some patients treated this way subsequently developed leukaemia, which most likely arises as a result of random insertion of a section of DNA into the human genome with the consequent disruption of nearby gene function. Cloning cells and animals In genetic engineering, the term “cloning” is now more commonly applied to the production of identical animals rather than molecular cloning of DNA fragments. Whole cell or animal cloning occurs through the transfer of the nucleus of an adult cell into an enucleated egg. This can result in the reprogramming of the adult cell DNA to produce a cloned animal. In 1997, a sheep named Dolly was born at the Roslin Institute in Edinburgh. She was created from the nucleus of a cultured mammary gland cell from a Finn Dorset sheep that was fused to an egg cell from a Scottish Blackface ewe that had had its own nucleus removed. The fused cell was implanted into a different Scottish Blackface ewe, and following a normal pregnancy, Dolly, a Finn Dorset sheep, was born. Nuclear transfer has subsequently been applied to produce a range of cloned animals including cows, goats, pigs, mice, and cats. References 1. ^ Copeland NG, Jenkins NA, Court DL (October 2001). "Recombineering: a powerful new tool for mouse functional genomics". Nat. Rev. Genet. 2 (10): 769–79. doi:10.1038/35093556. PMID 11584293.
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