Biological Sciences
In Vivo Cloning
In vivo cloning refers to the process of introducing foreign DNA into a living organism, such as a bacterial cell, to produce multiple copies of the DNA. This technique is commonly used in molecular biology to create large quantities of specific DNA sequences for further study or manipulation. In vivo cloning allows researchers to replicate and study genes and their functions within living organisms.
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5 Key excerpts on "In Vivo Cloning"
eBook - ePub- Boriana Marintcheva(Author)
- 2017(Publication Date)
- Academic Press(Publisher)
Principles of Molecular Cloning Cloning is the biological process of creating clones of genetically identical organisms, cells, or molecules. Clones of organisms arise via asexual reproduction. For example, each bacterial colony represents a clone comprised of the progeny of a single cell. Spider plants originating from clippings of the same plant are also considered clones. DNA cloning is the process of generating multiple copies of genetically identical DNA molecules, which usually involves insertion of a donor DNA fragment into a vector resulting in a recombinant DNA molecule, which is propagated using E. coli (Fig. 2.2). A classical cloning protocol involves (1) digestion of the donor and vector DNA to create compatible ends, (2) ligation, (3) transformation in competent E. coli cells, and (4) selection and verification of recombinant DNA molecules. Once the desired clone is selected, needed quantities of the recombinant DNA molecule are obtained by growing larger scale bacterial culture of E. coli harboring the molecule of interest. Essentially, the purpose of DNA cloning is to create a DNA clone, which is easy to manipulate and store for future purposes such as sequencing of the donor DNA fragment, mutagenesis, and protein expression. Often the end goal of DNA cloning is the generation of a DNA library, a collection of different DNA clones harboring unique DNA donor fragments cloned in the same vector using identical cloning protocol. The experimental steps are essentially the same as the ones for creating a single clone; however, they are performed simultaneously for multiple donor DNA fragments producing multiple clones
eBook - ePub- Firdos Alam Khan(Author)
- 2020(Publication Date)
- CRC Press(Publisher)
PCR has become a standard tool in forensic science because it can multiply very small samples of DNA for multiple crime lab testing. PCR has also become useful for archaeologists studying the evolutionary biology of different animal species, including samples thousands of years old. Cloning technology has made it relatively easy to isolate DNA fragments that contain genes to study gene function. Scientists believe that reliable cloning can be used to make farming more productive by replicating the best animals and crops and to make medical testing more accurate by providing test animals that all react the same way to the same drug. PCR is a revolutionary technology and is more efficient than gene cloning as it needs much less of the desired DNA (a single copy is enough). It is not difficult to store and does not require costly restriction enzymes, ligase, and vector DNA, thus drastically reducing the experimental cost. It needs far less work, time, and skill, and has many more applications than gene cloning. Typically, gene cloning experiments take 2–4 days, whereas PCR takes only up to 4–5 h. In addition, PCR is fully automated but gene cloning is not. Nevertheless, for PCR, one does need sequence information for construction of primers and a thermal cycler or PCR machine. It is expected that PCR will eventually take over most of the applications of gene cloning and will find many novel applications as well.4.7 SIGNIFICANCE OF VECTORS IN RECOMBINANT DNA TECHNOLOGY
Recall that in molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in a cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are called transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multi-cloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector, which transfers genetic information to another cell, is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (or expression constructs) are specifically for the expression of the transgene in the target cell and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated. They can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert. Insertion of a vector into the target cell is generally called transfection (Figure 4.6
eBook - ePub- Matt Carter, Jennifer C. Shieh(Authors)
- 2015(Publication Date)
- Academic Press(Publisher)
The essential features of a vector are that it can replicate autonomously in a host species, usually bacteria, and that it can be combined with other pieces of DNA. Often, the inserted DNA fragment comes from a different organism than the vector DNA, and the fusion is called a recombinant DNA construct. Two commonly used vectors are plasmids, naturally occurring circles of DNA that act as independently replicating accessory chromosomes in bacteria, and bacteriophage or phage, a virus that can deliver its genetic cargo to bacteria. It is much more common for scientists performing recombinant DNA techniques in the laboratory to use plasmids over phage vectors. Many plasmids have been ingeniously modified to enhance the delivery of recombinant DNA molecules into bacteria and to facilitate the selection of bacteria harboring these vectors. BOX 10.2 The Definition of Clone The word clone is used often in molecular biology. Unfortunately, this word has distinct definitions that can often cause confusion. In general, the precise definition of a clone is an exact copy. In reference to a piece of DNA, this term implies that a scientist will make exact copies of a DNA sequence. In reference to a bacterial cell, this term implies that a bacterium will multiply to eventually produce millions of genetically identical bacteria, each harboring the same DNA as the initial cell. Nowadays, it is possible to produce clones of larger organisms, including mammals—meaning that the animals share identical copies of genomic DNA. The term “cloning a gene” usually refers to the process of isolating a particular DNA sequence, placing the sequence in a vector, and producing many copies of the vector for use in subsequent experiments
eBook - ePub- Firdos Alam Khan(Author)
- 2018(Publication Date)
- CRC Press(Publisher)
4.7.1.2 How works DNA cloning involves first isolating the source and vector DNAs and then using enzymes to cut these two DNAs. Next, scientists bond the source DNA to the vector with a DNA ligase enzyme that repairs the splice and creates a single DNA strand. That DNA is then introduced into a host organism cell, where it grows with the organism.PCR has become a standard tool in forensic science because it can multiply very small samples of DNA for multiple crime lab testing. PCR has also become useful for archaeologists to study the evolutionary biology of different animal species, including samples thousands of years old. Cloning technology has made it relatively easy to isolate DNA fragments that contain genes to study gene function. Scientists believe that reliable cloning can be used to make farming more productive by replicating the best animals and crops and also to make medical testing more accurate by providing test animals that all react the same way to the same drug. PCR is a revolutionary technology and is more efficient than gene cloning, as it needs much less amount of the desired DNA (a single copy is enough). It is not difficult to store and does not require costly restriction enzymes, ligase, and vector DNA, thus reducing experimental cost drastically. It needs far less work, time, and skill and has many more applications than gene cloning. Typically, gene cloning experiments take 2–4 days, while PCR takes only up to 4–5 h. In addition, PCR is fully automated, while gene cloning is not. Nevertheless, for PCR, one does need sequence information for construction of primers and a thermal cycler or PCR machine. It is expected that PCR will eventually take over most of the applications of gene cloning and will find many novel applications as well.4.7.1.3 What are the differences between PCR and cloning?4.8 Significance of vectors in recombinant DNA technology
Recall that in molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are called transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multi-cloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector, which transfers genetic information to another cell, is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (or expression constructs) are specifically for the expression of the transgene in the target cell and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are capable of being only transcribed but not translated. They can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert. Insertion of a vector into the target cell is generally called transfection
eBook - ePubGenetic Engineering Fundamentals
An Introduction to Principles and Applications
- John Kammermeyer(Author)
- 2017(Publication Date)
- CRC Press(Publisher)
replicon. Usually, DNA fragments, which are to be cloned for the specific gene that they carry, do not contain a replicon. At this point the recombinant procedure comes into play. The DNA fragment is inserted into a plasmid or phage, as these entities contain their own replicons and so become “DNA carriers” or vectors. The engineered vectors are then inserted into a microbial host which recognizes the vector’s replicon. From here on the cloning procedure begins, that is the formation of new microbial entities which are identical to the original structure. Some foreign DNA fragments of course, can contain replicons which may be recognized by the host.THE CLONING PROCESSGene Introduction. The insertion of a specific DNA sequence into a vector, that is, the carrier of the new gene, requires the following conditions (6 ):1. A DNA vehicle (vector or replicon) which can replicate after foreign DNA is inserted in it. 2. A DNA molecule to be replicated: the foreign DNA insert, the passenger. 3. A method for introducing a passenger into the vehicle.4. A means for introducing the vehicle carrying the passenger into a host organism in which it can replicate; DNA transformation or transfection (the term transfection is used frequently with viral DNA).5. A means for screening or genetic selection for those cells that have replicated the desired recombinant molecules. This screening or selection process provides a way to recover the specific recombinant DNA in pure form. (The screening process by means of antibiotic resistances was illustrated in Figure 3 .)Elaboration:1. Cloning vehicles. Frequently used vehicles are bacterial plasmids from E. coli and B. subtilis; also used are yeast, streptomyces, bacteriophage λ, phage M13, and SV40 virus.2. DNA to be replicated. DNA fragments having the desired genome can be prepared by mechanical chopping (nonspecific fragments) or by digestion with restriction endonuclease (specific site cleavage). For a particular gene selection, the fragmented DNA is separated by means of gel electrophoresis (e.g., agarose gel). The fractions of the desired size are excised from the gel slab and eluted.Chemical synthesis of short segments (~ 40 bp) of oligodeoxynucleotides has been developed. Enzymatic joining of such segments to form double-stranded DNA containing synthetic genes is possible (see section on Synthesis). Thus, a number of desirable genes can be synthesized.
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