DNA Cloning

10 Key excerpts on "DNA Cloning"

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  eBook - ePub

  Molecular Biology and Genomics

  • Cornel Mulhardt(Author)
  • 2010(Publication Date)
  • Academic Press

    (Publisher)

  6

  Cloning DNA Fragments

  Publisher Summary

  Cloning is the introduction of a deoxyribonucleic acid (DNA) fragment into a vector, which makes it possible to increase this DNA to an abundant quantity. Cloning involves digesting the vector and DNA fragments, purifying them, ligating them with one another, and transforming the wild mixture that emerges within the bacteria. The cloning procedure is completed once the desired clone among the bacteria has been selected. The difficulties lie in the details. The decision about the right vector is one-half of the battle in cloning. The most difficult step is to find a suitable cutting site for cloning. It is recommended to use about five times more DNA fragments than the quantity of the vector. Quantity refers to calculation of the number of molecules, not the amount in nanograms. Anything can go wrong in cloning: no colonies may be obtained, or none of the clones may contain the desired insert. There could be various reasons that a cloning process does not work. For example, the ligase can give up the ghost. This possibility can be examined with the aid of a simple test by ligating 1 g of digested DNA (e.g., molecular-weight marker DNA without any blue marker) for 30 minutes. The largest part of the DNA should appear in the agarose gel as a high-molecular-mass smear.

   

  Des Löwen Mut, des Hirsches Schnelligkeit, des Italieners feurig Blut,

  des Nordens Dau’rbarkeit .

  The lion’s dauntless mood, The deer’s swift pace, The Italian’s fiery blood,

  The toughness of the northern race .

  The key word cloning should not to be confused with producing clones

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  eBook - ePub

  From Genes to Genomes

  Concepts and Applications of DNA Technology

  • Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant(Authors)
  • 2011(Publication Date)
  • Wiley

    (Publisher)

  2 How to Clone a Gene 2.1 What is cloning? Cloning is defined as the use of asexual reproduction to obtain organisms that are genetically identical to one another, and to the ‘parent’. This contrasts with sexual reproduction, where the offspring are not usually genetically identical. It is worth stressing that clones are only identical genetically ; the actual appearance and behaviour of the clones will be influenced by other factors such as their environment. This applies to all organisms, from bacteria to humans. Despite the emotive language surrounding the word ‘cloning’, this is a surprisingly familiar concept. In particular, anyone with an interest in gardening will know that it is possible to propagate plants by taking cuttings. These are clones. Similarly, the routine bacteriological procedure of purifying a bacterial strain by picking a single colony for inoculating a series of fresh cultures is also a form of cloning. The term cloning is applied to genes, by extension of the concept. If you introduce a foreign gene into a bacterium, or into any other type of cell, in such a way that it will be copied when the cell replicates, then you will produce a large number of cells all with identical copies of that piece of DNA – you have cloned the gene (Figure 2.1). By producing numerous copies in this way, you can sequence it or label it as a probe to study its expression in the organism it came from. You can express its protein product in bacterial or eukaryotic cells. You can mutate it and study what difference that mutation makes to the properties of the gene, its protein product, or the cell that carries it. You can even purify the gene from the bacterial clone and inject it into a mouse egg, and produce a line of transgenic mice that express it

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  eBook - PDF

  Human Heredity

  Principles and Issues

  • Michael Cummings(Author)
  • 2015(Publication Date)
  • Cengage Learning EMEA

    (Publisher)

  coli . The cloned DNA molecules can be recovered from the host cells and purified for further use. 13-4 Cloned Libraries ■ A collection of cloned DNA sequences from one source is a library. The clones in the library are a resource for work on specific genes. 13-5 Finding a Specific Gene in a Library ■ Clones for specific genes can be recovered from a library by using probes to screen the library. 13-6 A Revolution in Cloning: The Polymerase Chain Reaction ■ PCR is used to make many copies of a DNA molecule with-out using restriction enzymes, vectors, or host cells. It is faster and easier than conventional cloning. 13-7 Analyzing Cloned Sequences ■ Cloned sequences are characterized in several ways, including South-ern blotting and DNA se-quencing. 13-8 DNA Microarrays Are Used to Analyze Gene Expression ■ DNA microarrays can be used to analyze the expression of thousands of genes in a single experiment. This technology has a wide range of uses in genomics. Summary David Parker/Science Source Le Corre-Ribeiro/Liaison/Getty Images Michael Gabridge/Custom Medical Stock Courtesy of Affymetrix, Inc., Santa Clara, CA Corbin17/Alamy Copyright 2016 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. Due to electronic rights, some third party content may be suppressed from the eBook and/or eChapter(s). Editorial review has deemed that any suppressed content does not materially affect the overall learning experience. Cengage Learning reserves the right to remove additional content at any time if subsequent rights restrictions require it. 304 Chapter 13 What Are Clones? 1. Cloning is a general term used for whole organisms and DNA sequences. Define what we mean when we say we have a clone. 2. Nuclear transfer to clone cattle is done by which of the following techniques? a. An 8-cell embryo is divided into two 4-cell embryos and implanted into a surrogate mother.

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  eBook - ePub

  Guide to Research Techniques in Neuroscience

  • Matt Carter, Jennifer C. Shieh(Authors)
  • 2009(Publication Date)
  • Academic Press

    (Publisher)

  Though the primary purpose of PCR is to make exact copies of a DNA sequence, it can also be used to modify a sequence and make copies of the modified sequence. This is primarily done by creating primers that contain the desired sequence modifications, either by adding the base pairs of specific restriction enzyme recognition sites to the ends or by altering a single base pair to introduce a point mutation. Though the primers may not hybridize as tightly for the first few PCR cycles, with each successive cycle, the added or mutated sequences in the primers will be copied and incorporated into the amplified PCR product.

  Cloning DNA

  The core of molecular cloning is creating many copies of a particular DNA fragment (Box 9.2 ). In order to clone a DNA fragment that has been isolated using restriction enzyme digests or PCR, it must be put into a form that can be copied. Bacterial host cells are often used as factories to mass-produce clones of the DNA of interest. It is these colonies of identical bacterial clones that lend their name to the technique of “molecular cloning.” For bacteria to mass produce DNA of interest, it must be in a form that can be replicated and passed on by the dividing cells. Thus, isolated DNA fragments must be put into a storage device: the vector. If the DNA fragment comes from another vector, the process of transferring the desired sequence into a new vector is called subcloning . Box 9.5 describes an example of a subcloning experiment, going through all the steps that will be described in the following section.

  Box 9.2

  Defining the Word Clone

  The word clone has many definitions, which can make terminology in molecular biology rather confusing. The general definition involves making copies, but the term also describes distinct though related topics. Animal or cell clones are so called because of the shared copies of genomic DNA. Mapping the location of a gene discovered through a forward genetic screen (Chapter 8 ) is also known as positional cloning

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  eBook - ePub

  Essentials of Chemical Biology

  Structure and Dynamics of Biological Macromolecules

  • Andrew D. Miller, Julian Tanner, Julian A. Tanner(Authors)
  • 2013(Publication Date)
  • Wiley

    (Publisher)

  Direct DNA Cloning was originally developed for the isolation and cloning of prokaryotic genes but can be used for the isolation and cloning of eukaryotic genes as well. The meaning of direct DNA Cloning is the isolation and multiple copying of DNA from a source of interest (i.e. genomic DNA primarily purified directly from a prokaryotic organism, or DNA established in a eukaryotic cDNA library). With respect to prokaryotic organisms, DNA once isolated must be cut into fragments (‘shot-gun’ fragments) either by chemical means or by using restriction enzymes (see Section 3.2.2). Restriction fragments of genomic DNA are then ligated into pDNA vectors (see Section 3.2.1) to create recombinant pDNA constructs. With respect to eukaryotic organisms, cDNA library components may be inserted similarly into pDNA to create alternate eukaryotic recombinant pDNA constructs.

  Figure 3.13 Automated DNA sequencing. When ddNTPs are differentially fluorescent-labelled, then only a single template-directed DNA polymerization reaction is required spiked with all four ddNTPs. The different length oligodeoxynucleotide products are then resolved individually by capillary electrophoresis and the identity of the terminating ddNTP can be identified not by horizontal position on a gel (see Fig. 3.12 ) but by colour of extrinsic fluorescence output (red for ddTTP, blue for ddCTP, green for ddATP and cyan for ddGTP) (see Chapter 4). The collective sequence of fluorescence peaks together represent another version of the sequencing ladder that is read from left to right identifying the DNA sequence (5′ to 3′) (400-800 bases) complementary to the template DNA, reading directly from the selected primer (Reproduced from Voet, Voet & Pratt, 1999 [Wiley] Fig. 3-25).

  Recombinant means that the pDNA is comprised of regions of DNA from at least two different organisms. These recombinant pDNA constructs are used in the transformation of host cells, typically E. coli . Multiple copying of the recombinant pDNA constructs then becomes possible with growth of the host owing to simple DNA replication. Since the genetic code is universal, the host is unable to distinguish recombinant DNA from self- (host) DNA, and consequently, when the host grows and divides, even the recombinant pDNA is copied. If E. coli cells are spread on a nutrient plate and allowed to grow, then colonies form such that each cell of a given colony originates from a single parent cell and is a clone of the original transformed cell. Each cell of the colony will possess recombinant pDNA (as single plasmid or multiple copy/clonal cell) containing a single, unique restriction fragment not found in any of the other E. coli colonies. The result then is known as a clonal library,

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  eBook - ePub

  BIOS Instant Notes in Biochemistry

  • David Hames, Nigel Hooper(Authors)
  • 2011(Publication Date)
  • Taylor & Francis

    (Publisher)

  vector.

  A population of recombinant DNA molecules can be made, each recombinant molecule containing one of the mouse DNA fragments in the original mixture. This can then be introduced into a population of bacteria such that each bacterial cell contains, in general, a different type of recombinant DNA molecule. If we can identify the bacterial cell that contains the recombinant DNA bearing the mouse DNA fragment we want, the cell can be multiplied in culture and large amounts of the recombinant DNA isolated. The mouse DNA of interest can then be recovered from this in pure form; it is then said to have been cloned.

  The vector that was used to achieve this cloning is called a cloning vector. Vectors are not limited to bacterial cells; animal and plant viruses can also act as vectors.

  The basics of DNA Cloning

  There are a wide variety of different procedures for cloning DNA into either plasmid or viral vectors and the reader is referred to the companion textbook Instant Notes in Molecular Biology for more detail. However, the basic scheme of events is often the same. To clone into a plasmid vector, the circular plasmid DNA is cut with a restriction enzyme (Section I2) that has only a single recognition site in the plasmid. This creates a linear plasmid molecule with cohesive ends (Figure 1 ). The simplest cloning strategy is now to cut the DNA that is to be cloned (in the above example, mouse DNA) with the same restriction enzyme. Alternatively, different restriction enzymes can be used, provided they create the same cohesive ends (Section I2). The restricted mouse DNA and linear plasmid DNA are now mixed. The cohesive ends of the mouse DNA anneal with the ends of the plasmid DNA and are joined covalently by DNA ligase. The resulting recombinant plasmid DNA is introduced into bacterial host cells that have been treated to become permeable to DNA. This uptake of DNA by the bacterial cells is called transfection; the bacterial cells are said to have been transfected

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  eBook - ePub

  Genomics and Clinical Diagnostics

  • David Whitehouse, Ralph Rapley(Authors)
  • 2019(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  Many of these have been through the isolation and characterisation of numerous DNA-manipulating enzymes, such as DNA polymerase, DNA ligase and reverse transcriptase, for example. However, perhaps one of the most important was the isolation and application of enzymes that enabled the reliable and reproducible digestion of DNA. These enzymes, termed restriction endonucleases, not only provided a turning point for the analysis of DNA but also provided the means to develop recombinant DNA technology as well as key methods for gene library production, molecular cloning, DNA sequencing and also gene editing. 3.1.1 The Molecular Cloning Process Molecular cloning provides a convenient way of dividing the very large segments of DNA associated with genomes into smaller pieces that are suited to detailed analysis. 1 Cloning involves the amplification of specific segments of DNA, leading to the accumulation of large amounts of identical DNA fragments that can be used for various purposes. In many ways the process is analogous to amplification of DNA by PCR. Classical molecular cloning employs bespoke DNA Cloning vectors, which are often derived from circular DNA molecules called bacterial plasmids and bacteriophages, such recombinant vectors replicate efficiently in specialised strains of bacterial host cells, resulting in the production of multiple copies of the recombinant DNA molecule that was inserted into the vector. Before a DNA fragment can be inserted into a cloning vector, it must be prepared so that the precise DNA sequences of its ends are compatible with the ends of the vector molecule DNA sequence. There are ways to produce recombinant molecules independent of this process but cell-based cloning still remains the method of choice for most situations. Cell-based cloning is achieved using commercially available enzymes that cleave double stranded DNA at specific sequences; these are known as restriction enzymes or restriction endonucleases (see Chapter 1)

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  eBook - ePub

  Genetic Engineering

  Volume 1: Principles Mechanism, and Expression

  • Tariq Ahmad Bhat, Jameel M. Al-Khayri, Tariq Ahmad Bhat, Jameel M. Al-Khayri(Authors)
  • 2023(Publication Date)
  • Apple Academic Press

    (Publisher)

  There are various types of cloning vectors with respect to the size of desired gene which is to be inserted in cloning vector. A brief list of such cloning vectors is given in subsections.

  5.2.1 Plasmids

  Plasmid is a small, autonomously-replicating extra-cellular DNA molecule which is present in the cell, besides chromosomal DNA. They are mostly found in bacteria whereas, some archaea as well as eukaryotes are also known to have plasmids. These are the most commonly used cloning vectors for the synthesis of recombinant cells. Plasmids have the capacity to efficiently clone approx. 10 kb of gene of interest. Any gene below this size can easily be cloned and manipulated using this system however, plasmids are not efficient to clone larger size DNA inserts (Lodish & Zipursky, 2001 ). Bacteria are often used as host cells for plasmids; however, cell lines can also be transformed using plasmids as vectors. Copy number is a term referred to the number of copies of plasmids per cell. A plasmid copy number is directly related to the structural characteristics of its Ori point. Other contributing factors include, plasmid size and culture condition for cell growth. All these factors can be manipulated in order to design a plasmid with desired properties. pUC 19 is a genetically engineered (GE) cloning vector with high copy number (Figure 5.6 ). Its Ori point is manipulated in such a way as to produce 500–700 copies of plasmids per cell.

  Figure 5.6 Illustration of pUC19 vector.

  Source: Adapted from Boca Scientific Inc.

  5.2.2 Bacteriophage

  Bacteriophage is a virus that eats on bacteria which are have various applications in rDNA technology. M13 phage and λ phage are the most frequently used bacteriophages in cloning. Phages usually contain a linear DNA molecule and unlike plasmids, single cut in DNA strand will generate two fragments. These fragments can subsequently be fused with foreign DNA elements to generate a chimeric phage. For synthesizing these chimera, foreign DNA can be fetched either from another phage or a plasmid donor carrying the gene of interest as depicted in Figure 5.7 (Chen et al., 2019

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  eBook - ePub

  Genetic 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 PROCESS

  Gene 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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  Human Cloning in the Media

  From Science Fiction to Science Practice

  • Joan Haran, Jenny Kitzinger, Maureen McNeil, Kate O'Riordan(Authors)
  • 2007(Publication Date)
  • Routledge

    (Publisher)

  2 What is cloning?

  We should not see cloning as an isolated technology, single-mindedly directed at replication of livestock or of people. It is the third player in a trio of modern biotechnologies that have arisen since the early 1970s. Each of the three, taken alone, is striking; but taken together, they propel humanity into a new age – as significant, as time will tell, as our forebear’s transition into the age of steam, or of radio, or of nuclear power (Wilmut et al. 2000a: 6).

  The point is that the three technologies together – genetic engineering, genomics and our method of cloning from cultured cells – are a very powerful combination (ibid.: 9).

  Introduction

  Ian Wilmut and his colleagues make dramatic claims for cloning as a technology. However, cloning captured popular attention long before news of the birth of Dolly the sheep in 1997 and before the announcement of the ‘completion’ of the Human Genome Project in 2000. Nevertheless, in the aftermath of these two announcements there has been a renewed and intensified interest in cloning. This chapter brings the contemporary concern with human cloning under scrutiny both by locating it in a set of genealogies and by analysing the distinctive features of its recent manifestations. (See Appendix I for a timeline that attempts to consolidate these genealogies to provide an overview of key events.) In this chapter, we draw on a range of accounts by scientists and social commentators on the development of the technology of cloning and provide a brief review of some of its key representations in Western popular culture. This sets the context for our analysis of the particular configurations of cloning discourses that have become dominant in the early twenty-first century.

  It is tempting at this point to offer a dictionary definition of cloning as our starting point. For example, the Chambers Dictionary

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