Cloning Amplifies DNA
المؤلف:
Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil
المصدر:
Harpers Illustrated Biochemistry
الجزء والصفحة:
32nd edition.p447-449
2025-10-23
106
A clone is a large population of identical molecules, cells, or organisms that arise from a common ancestor. Molecular cloning allows for the production of a large number of identical DNA molecules, which can then be characterized or used for other purposes. This technique is based on the fact that chimeric or hybrid DNA molecules can be constructed in cloning vectors—typically bacterial plasmids, phages, or cosmids (hybrid plasmids that also contain specific phage sequences)—which then continue to replicate clonally in a single host cell under their own control systems. In this way, the chimeric DNA is amplified. The general procedure is illustrated in Figure 1.
Fig1. Use of restriction endonucleases to make new recombinant or chimeric DNA molecules. When inserted back into a bacterial cell (by the process called DNA-mediated transformation), typically only a single plasmid is taken up by a single cell, and the plasmid DNA replicates clonally, not only itself, but also the physically linked new DNA insert. Since recombining the sticky ends, as indicated, typically regenerates the same DNA sequence recognized by the original restriction enzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this endonuclease. Alternatively, the insert sequences can be specifically amplified from the purified chimeric plasmid DNA by PCR. If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restriction nuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules can be obtained, each pure in its own bacterial clone.
Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA as episomes (ie, a genome above or outside the bacterial genome) while using primarily the host replication machinery. The complete DNA sequence of thousands of plasmids is known; hence, the precise location of restriction enzyme cleavage sites for inserting foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily biochemically separated from the latter, and the desired plasmid-inserted DNA can be readily removed by cutting the plasmid with the enzyme specific for the restriction site into which the original piece of DNA was inserted.
Phages (bacterial viruses) often have linear DNA genomes into which foreign DNA can be inserted at unique restriction enzyme sites. The resulting chimeric DNA is collected after the phage proceeds through its lytic cycle and produces mature, infective phage particles. A major advantage of phage vectors is that while plasmids accept DNA pieces up to about 10-kb long, phages can readily accept DNA fragments up to ~20-kb long. The ultimate insert size is imposed by the amount of DNA that can be packed into the phage head during virus propagation.
Larger fragments of DNA can be cloned in cosmids, DNA cloning vectors that combine the best features of plasmids and phages. Cosmids are plasmids that contain the DNA sequences, so-called cohesive end sites (cos sites), required for packaging lambda DNA into the phage particle. These vectors grow in the plasmid form in bacteria, but since much of the unnecessary lambda DNA has been removed, more chimeric DNA can be packaged into the particle head. Cosmids can carry inserts of chimeric DNA that are 35- to 50-kb long. Even larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or E. coli bacteriophage P1-derived artificial chromo some (PAC) vectors. These vectors will accept and propagate DNA inserts of several hundred kilobases or more, and have largely replaced the plasmid, phage, and cosmid vectors for some cloning and eukaryotic gene mapping/expression applications. A comparison of these vectors is shown in Table 1.
Table1. Cloning Capacities of Common Cloning Vectors
Because insertion of DNA into a functional region of the vector will interfere with the action of this region, care must be taken not to interrupt an essential function of the vector. This concept can be exploited, however, to provide a powerful double positive/negative selection technique. For example, a common early plasmid vector pBR322 has genes conferring resistance to both tetracycline (Tet) and ampicillin (Amp), that is, Tetr- and Ampr-resistant growth, respectively. A single PstI restriction enzyme site within the Amp resistance gene is commonly used as the insertion site for a piece of foreign DNA. In addition to having sticky ends, the DNA inserted at this site disrupts the ORF of the β-lactamse-encoding bla gene. β-Lactamase, a secreted enzyme degrades and inactivates ampicillin. A bacterium carrying a plasmid with an inserted DNA fragment in the bla gene will be Amp-sensitive (Amps). Thus, cells carrying the parental plasmid, which provides resistance to both antibiotics, can be readily distinguished via replica plating, and separated from cells carrying the chimeric plasmid, which is resistant only to tetracycline (Figure 2). YACs contain selection, replication, and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in either organism.
Fig2. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. This insertion disrupts the codon reading frame for the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, cells carrying the chimeric plasmid will no longer grow/survive when cultured in liquid, or plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying on production of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an inactive, N-terminally truncated form of the enzyme β-galactosidase, a component of the lac operon allows for blue–white colony formation on agar plates containing a dye hydrolyzable by β-galactosidase. β-galactosidase–positive colonies are blue; such colonies contain plasmids in which a DNA was successfully inserted.
In addition to the vectors described in Table 1 that are designed primarily for propagation in bacterial cells, vectors for mammalian cell propagation and insert gene-(cDNA)/ protein expression have also been developed. These vectors are all based on various eukaryotic viruses that are composed of RNA or DNA genomes. Notable examples of such viral vectors are those utilizing adenoviral (Ad), or adenovirus-associated viral (AAV)(DNA-based) andretroviral(RNA based) genomes. Though somewhat limited in the size of DNA sequences that can be inserted, suchmammalian viral cloning vectorsmake up for this shortcoming because they will efficiently infect a wide range of different cell types. For this reason, various mammalian viral vectors, some with both positive and negative selection genes (aka selectable “markers” as noted earlier for pBR322) are being used for both laboratory experiments and human gene therapy.
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