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You are watching: Describe the construction of a recombinant plasmid

Griffiths AJF, müller JH, Suzuki DT, et al. An introduction to genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.


By covenant with the publisher, this publication is available by the search feature, yet cannot be browsed.
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How walk recombinant DNA modern technology work? The biology under study, which will beused to donate DNA because that the analysis, is dubbed the donor organism. Thebasic procedure is come extract and cut up DNA indigenous a donor genome right into fragmentscontaining native one to number of genes and permit these pieces to insert themselvesindividually right into opened-up little autonomously replicating DNA molecules such asbacterial plasmids. These little circular molecules act together carriers, orvectors, for the DNA fragments. The vector molecules with theirinserts are called recombinant DNAbecause lock consist of novel combinations of DNA native the donor genome (which canbe from any organism) with vector DNA native a completely different resource (generallya bacterial plasmid or a virus). The recombinant DNA mixture is then offered totransform bacter cells, and also it is typical for solitary recombinant vector moleculesto uncover their method into individual bacterial cells. Bacterial cells are plated andallowed to grow into colonies. One individual revolutionized cell v a singlerecombinant vector will divide right into a colony with numerous cells, every carryingthe same recombinant vector. Therefore an individual colony contains a very largepopulation of the same DNA inserts, and this populace is referred to as a DNA clone. A an excellent deal that the analysisof the copy DNA fragment deserve to be performed at the stage when it is in the bacterialhost. Later, however, that is often desirable to reintroduce the copy DNA earlier intocells the the original donor organism to lug out details manipulations the genomestructure and function. Therefore the protocol is often as follows:

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MESSAGE

Cloning enables the amplification and recovery of a details DNA segment indigenous alarge, complicated DNA sample such as a genome.


Inasmuch together the donor DNA was cut into many different fragments, most nests willcarry a various recombinant DNA (that is, a different cloned insert). Therefore,the next step is to uncover a way to choose the clone v the insert containing thespecific gene in i m sorry we space interested. As soon as this clone has actually been obtained, the DNAis diverted in bulk and the copy gene the interest deserve to be subjected to a variety ofanalyses, which us shall consider later in the chapter. Notice that the cloningmethod works since individual recombinant DNA molecules go into individual bacterialhost cells, and then these cells perform the job of amplifying the single molecules intolarge populaces of molecules that can be treated as chemical reagents. Number 12-1 provides a general outline of theapproach.


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Figure 12-1

Recombinant DNA technology enables individual pieces of DNA from anygenome to be inserted into vector DNA molecules, such as plasmids, andindividually enhanced in bacteria. Each enhanced fragment is referred to as aDNA clone.


The hatchet recombinant DNA must be identified from the organic DNArecombinants that result from crossing-over between homologous chromosomes in botheukaryotes and prokaryotes. Recombinant DNA in the sense being used in this chapteris an unnatural union of DNAs from nonhomologous sources, normally from differentorganisms. Some geneticists prefer the alternative name chimeric DNA, ~ the superordinary Greek monster Chimera.Through the ages, the Chimera has actually stood as the price of an impossible biologicalunion, a mix of parts of different animals. Likewise, recombinant DNA is aDNA chimera and also would be difficult without the speculative manipulation that wecall recombinant DNA technology.


Isolating DNA

The an initial step in making recombinant DNA is to isolation donor and vector DNA.General protocols for DNA isolation were obtainable many decades before theadvent that recombinant DNA technology. V the use of together methods, the bulk ofDNA extracted from the donor will be atom genomic DNA in eukaryotes or themain genomic DNA in prokaryotes; these types are normally the ones forced foranalysis. The procedure used for obtaining vector DNA depends on the nature ofthe vector. Bacterial plasmids are typically used vectors, and these plasmidsmust it is in purified far from the bacter genomic DNA. A protocol for extractingplasmid DNA through ultracentrifugation is summarized in number 12-2. Plasmid DNA develops a unique band afterultracentrifugation in a cesium chloride thickness gradient containing ethidiumbromide. The plasmid tape is built up by punching a hole in the plasticcentrifuge tube. One more protocol relies on the observation that, at a specificalkaline pH, bacterial genomic DNA denatures yet plasmids do not. Subsequentneutralization precipitates the genomic DNA, yet plasmids remain in solution.Phages such as λ additionally can be supplied as vectors because that cloning DNA in bacterialsystems. Phage DNA is isolated native a pure suspension of phages recovered from aphage lysate.


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Figure 12-2

Plasmids such together those carrying genes because that resistance to theantibiotic tetracycline (top left) have the right to be separatedfrom the bacter chromosomal DNA. Because differential binding ofethidium bromide by the 2 DNA types makes the circular plasmidDNA (more...)


Cutting DNA

The breakthrough that made recombinant DNA technology feasible was the discoveryand characterization the restriction enzymes. limit enzymesare developed by bacteria together a defense mechanism against phages. The enzymes actlike scissors, cutting up the DNA the the phage and thereby inactivating it.Importantly, restriction enzymes execute not cut randomly; rather, they reduced atspecific DNA target sequences, which is among the crucial features that make themsuitable for DNA manipulation. Any kind of DNA molecule, from viral to human, containsrestriction-enzyme target sites completely by chance and therefore might be cut intodefined pieces of a size suitable for cloning. Restriction sites space notrelevant come the role of the organism, and they would not be cut in vivo,because many organisms do not have restriction enzymes.

Let’s look in ~ an example: the limit enzyme EcoRI (fromE. Coli) recognizes the adhering to six-nucleotide-pairsequence in the DNA of any organism:

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This kind of segment is referred to as a DNA palindrome, which way thatboth strands have actually the exact same nucleotide sequence however in antiparallel orientation.Many various restriction enzyme recognize and cut specific palindromes. Theenzyme EcoRI cut within this sequence however in a pair ofstaggered cuts between the G and the A nucleotides.

This staggered cut leaves a pair of similar single-stranded “sticky ends.” Theends are called sticky due to the fact that they have the right to hydrogen shortcut (stick)to a security sequence. Number 12-3shows EcoRI do a solitary cut in a one DNA molecule suchas a plasmid: the cut opens increase the circle, and also the linear molecule created hastwo sticky ends. Manufacturing of these sticky ends is another feature ofrestriction enzyme that makes them perfect for recombinant DNA technology. Theprinciple is merely that, if two various DNA molecules are reduced with the samerestriction enzyme, both will produce fragments v the very same complementarysticky ends, do it feasible for DNA chimeras come form. Hence, if both vectorDNA and also donor DNA are cut with EcoRI, the difficult ends of thevector have the right to bond come the difficult ends the a donor fragment once the two aremixed.


Figure 12-3

The border enzyme EcoRI cut a circular DNAmolecule bearing one target sequence, bring about a straight moleculewith single-stranded sticky ends.


MESSAGE

Restriction enzymes have two properties useful in recombinant DNA technology.First, they reduced DNA into fragments of a size perfect for cloning. Second,many border enzymes do staggered cuts that produce single-strandedsticky ends conducive come the formation of recombinant DNA.


Dozens of restriction enzyme with different sequence specificities have now beenidentified, several of which are shown in Table12-1. Friend will an alert that all the target sequences room palindromes,but, prefer EcoRI, some enzymes do staggered cuts, whereasothers do flush cuts. Also flush cuts, which absence sticky ends, can be used formaking recombinant DNA.


DNA can additionally be cut by mechanical shearing. Because that example, agitating DNA in ablender will certainly break increase the lengthy chromosome-sized molecules right into flush-endedclonable segments.


Joining DNA

Most commonly, both donor DNA and also vector DNA room digested with the usage of arestriction enzyme that produces difficult ends and also then combined in a test pipe toallow the difficult ends of vector and donor DNA to bind to every other and formrecombinant molecules. Number 12-4ashows a plasmid vector that carries a solitary EcoRI restrictionsite; therefore digestion through the restriction enzyme EcoRI convertsthe circular DNA right into a direct molecule with sticky ends. Donor DNA from anyother resource (say, Drosophila) likewise is treated with theEcoRI enzyme to develop a populace of fragments carryingthe exact same sticky ends. Once the two populaces are mixed, DNA pieces from thetwo sources can unite, because twin helices kind between their difficult ends.There are countless opened-up vector molecule in the solution, and many differentEcoRI pieces of donor DNA. As such a diverse selection ofvectors carrying various donor inserts will be produced. At this stage,although sticky ends have united to generate a population of chimeric molecules,the sugar-phosphate backbones space still not complete at 2 positions in ~ eachjunction. However, the backbones have the right to be sealed by the addition of the enzymeDNA ligase, which develop phosphodiester bonds in ~ the junctions(Figure 12-4b). Certain ligases areeven capable of authorized DNA fragments with blunt-cut ends.


Figure 12-4

Amplifying recombinant DNA

The ligated recombinant DNA start a bacterial cabinet by transformation. After ~ itis in the host cell, the plasmid vector is able to replicate since plasmidsnormally have actually a replication origin. However, currently that the donor DNA insert ispart that the vector’s length, the donor DNA is immediately replicated with the vector. Each recombinant plasmid the enters a cabinet will kind multiplecopies of itself in the cell. Subsequently, numerous cycles the cell division willtake place, and also the recombinant vectors will undergo much more rounds that replication.The resulting colony of bacteria will contain billions of copies of the singledonor DNA insert. This collection of amplified copies that the single donor DNA fragmentis the DNA clone (Figure 12-5).

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Figure 12-5

How amplification works. Restriction-enzyme treatment of donor DNAand vector permits insertion of single fragments into vectors. Asingle vector enters a bacterial host, where replication and also cell division result in a huge number of duplicates of the donorfragment. (more...)


By covenant with the publisher, this book is available by the find feature, however cannot be browsed.