Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. Molecular cloning generally uses DNA sequences from two different organisms: Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments.
The recombinant DNA is then introduced into a host organism typically an easy-to-grow, benign, laboratory strain of E. This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule.
Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental recombinant dna sentence examples used to assemble them.
The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes. Virtually any DNA sequence can be cloned and amplified, but there are some factors that recombinant dna sentence examples limit the success of the process.
Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. Another characteristic that limits chances of success is large size of DNA sequence.
Prior to the s, our understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleasesenzymes that could cleave DNA molecules only when specific DNA sequences were encountered.
Using a second enzyme, DNA ligasefragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures.
The first recombinant DNA molecules were generated and studied in Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the recombinant dna sentence examples in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replicationand the resulting recombinant DNA is introduced into recombinant dna sentence examples organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism.
The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates Recombinant dna sentence examples in an in vitro solution, free of living cells. In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.
PCR primer design, dedicated software exist for the purpose. Notably, the growing capacity recombinant dna sentence examples fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These shifts recombinant dna sentence examples complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction.
Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. Specialized applications may call for specialized host-vector systems. For example, recombinant dna sentence examples the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism.
Alternatively, if replication of the DNA in different species is recombinant dna sentence examples for example, transfer of DNA from bacteria to plantsthen a multiple host range vector also termed shuttle vector may recombinant dna sentence examples selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a recombinant dna sentence examples plasmid, followed by subcloning into a specialized vector.
Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility: The cloning vector is treated with a restriction endonuclease recombinant dna sentence examples cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA see DNA end.
Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule so that the vector can only be cleaved at a single site and are located within a gene frequently beta-galactosidase whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process.
To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme alkaline phosphatase that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site. Virtually any tissue source can be used even tissues from extinct animals [12]as long as the DNA is not extensively degraded.
The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA linkers containing desired restriction sites may be added to create end structures that are compatible with the vector. The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an recombinant dna sentence examples DNA ligase that covalently links the ends together.
This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism. DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA molecules with randomly joined ends.
This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells. The Recombinant dna sentence examples mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process recombinant dna sentence examples often depend upon the experimental method that is chosen e.
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformationand cells that are in a physiological state such that they can take up DNA are said to be competent. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types recombinant dna sentence examples are used.
Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane and cell wall, if present. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.
Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in recombinant dna sentence examples cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
When bacterial cells are used as recombinant dna sentence examples organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid sequences will die. When mammalian cells e. Recombinant dna sentence examples bacterial cloning vectors e. In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidasean enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work.
Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing transformed DNA remain recombinant dna sentence examples white. Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.
Recombinant dna sentence examples total population of individual clones recombinant dna sentence examples in a molecular cloning experiment is often termed a DNA library. Libraries may be highly complex as when cloning complete genomic DNA from an organism or relatively simple as when moving a previously cloned DNA fragment into a different plasmidbut it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained.
Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from recombinant dna sentence examples genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science.
A few of the more important applications are summarized here. Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned fragments of the genome, and assembling the overlapping sequences.
At the level of individual genes, molecular clones are used to generate probes that are used for examining how genes are expressedand how that expression is related to other processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death.
Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate the genes, or make more subtle mutations using regional mutagenesis or site-directed mutagenesis.
Genes cloned into expression vectors for functional cloning provide a means to screen for genes on the basis of the expressed protein's function. Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein.
In practice, it is frequently more difficult to develop an organism recombinant dna sentence examples produces an active form of the recombinant protein in desirable quantities than it is to clone the gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.
Many useful proteins are currently available as recombinant products. These include-- 1 medically useful proteins whose administration can correct a defective recombinant dna sentence examples poorly expressed gene e. Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, recombinant dna sentence examples transgenic organisms, also termed genetically modified organisms GMOs.
Although most GMOs are generated for purposes of basic biological research see for example, transgenic mousea number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds pharmingherbicide-resistant crop plantsand fluorescent tropical fish GloFish for home entertainment.
Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or recombinant dna sentence examples 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. 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 s, mostly for the treatment of cancers and blood, liver, and lung disorders. Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success.
Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.
From Wikipedia, the free encyclopedia. Gene cloning and DNA analysis: Annual Review of Biochemistry. Cold Spring Harbor, N. Cold Spring Harbor Laboratory. DNA technology's first drug". American Journal of Hospital Pharmacy. Annals of Emergency Medicine. Taiwan Childhood Hepatoma Study Group". The New England Journal of Medicine.
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