Recombinant DNA Technology Involves Isolation & Manipulation of DNA to Make Chimeric Molecules

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                 Isolation and manipulation of DNA, including end-to-end joining of sequences from very different sources to make chimeric molecules (e.g., molecules containing both human and bacterial DNA sequences in a sequence-independent fashion), is the essence of recombinant DNA research . This involves several unique techniques and reagents.RESTRICTION ENZYMES CUT3 DNA CHAINS AT SPECIFICLOCATIONS Certain endonucleases ?aenzymes that cut DNA at specific DNA sequences within the molecule (as opposed to exonucleases , which digest from the ends of DNA molecules)?aare a key tool in recombinant DNA research. These enzymes were called restriction enzymes because their presence in a given bacterium restricted the growth of certain bacterial viruses called bacteriophages. Restriction enzymes cut DNA of any source into short pieces in a sequence-specific manner?ain contrastto most other enzymatic, chemical, or physical methods, which break DNA randomly. These defensive enzymes (hundreds have been discovered) protect the host bacterial DNA from DNA from foreign organisms (primarily infective phages). However, they are present only in cells that also have a companion enzyme which methylates the host DNA, rendering it an unsuitable substrate for digestion by the restriction enzyme. Thus, site-specific DNA methylases and restriction enzymes always exist in pairs in a bacterium. RESTRICTIONENZYMES & DNA LIGASE ARE USED TO PREPARE CHIMERIC DNA MOLECULES Sticky-end ligation is technically easy, but some special techniques are often required to overcome problems inherent in this approach. Sticky ends of a vector may reconnect with themselves, with no net gain of DNA. Sticky ends of fragments can also anneal , so that tandem heterogeneous inserts form. Also, sticky-end sites may not be available or in a convenient position. To circumvent these problems, an enzyme that generates blunt ends is used, and new ends are added using the enzyme terminal transferase. If poly d(G) is added to the 3' ends of the vector and poly d(C) is added to the 3' ends of the foreign DNA, the two molecules can only anneal to each other, thus circumventing the problems listed above. This procedure is called homopolymer tailing12. Sometimes, synthetic blunt-ended duplex oligonucleotide linkers with a convenient restriction enzyme sequence are ligated to the blunt-ended DNA. Direct blunt-end ligation is accomplished using the enzyme bacteriophage T4 DNA ligase. This technique, though less efficient than sticky-end ligation, has the advantage of joining together any pairs of ends. The disadvantages are that there is no control over the orientation of insertion or the number of molecules annealed together, and there is no easy way to retrieve the insert. CLONING AMPLIFIES DNA A clone is a large population of identical molecules, bacteria, or cells 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?atypically bacterial plasmids), phages, or cosmids?awhich then continue to replicate in a host cell under their own control systems. In this way, the chimeric DNA is amplified. The general procedure is illustrated in Figure 1. Fig.1 Use of restriction nucleases to make new recombinant or chimeric DNA molecules. When inserted back into a bacterial cell (by the process called transformation), typically only a single plasmid is taken up by a single cell, and the plasmid DNA replicates not only itself but also the physicallylinked new DNA insert. Since recombining the sticky ends, as indicated, 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. If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restrictionnuclease 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. (Modified and reproduced, with permission, from Cohen SN: The manipulation of genes. Sci Am [July] 1975; 233: 25. The Estate of Bunji Tagawa.) 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. The complete DNA sequence of many plasmids is known; hence, the precise location of restriction enzyme cleavage sites for inserting the foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily separated from the latter, and the desired plasmid-inserted DNA is readily removed by cutting the plasmid with the enzyme specific for the restriction site into which the original piece of DNA was inserted. Phages usually have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites. The 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 about 6¨C10 kb long, phages can accept DNA fragments 10¨C20 kb long, a limitation imposed by the amount of DNA that can be packed into the phage head. Larger fragments of DNA can be cloned in cosmids, which combine the best features of plasmids and phages. Cosmids are plasmids that contain the DNA sequences, so-called 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. It is not unusual for cosmids to carry inserts of chimeric DNA that are 35-50 kb long. Even larger pieces of DNA can be incorporatedinto bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or E. coil bacteriophage Pl-based (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 gene mapping applications. A comparison of these vectors is shown in Table 1. Table 1 Cloning Capacities of Common Cloning Vector. 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 selection technique. For example, the common plasmid vector pBR322 has both tetracycline (tet) and ampicillin (amp) resistance genes. A single PstI restriction enzyme site within the amp resistance gene is commonly used as the insertion site for a piece of foreign the DNA inserted at this site disrupts the amp resistance gene and makes the bacterium carrying this plasmid amp-sensitive (Fig. 2). Thus, the parental plasmid, which provides resistance to both antibiotics, can be readily separated from the chimeric plasmid, which is resistant only to tetracycline. YACs contain selection, replication, and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in either organism. Fig.2 A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is insertedinto the unique Pstl site. This inserting disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when 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 similarscheme relying upon production of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an inactive, deleted form of the enzyme |?-galactosidase allows for blue-white colony formation on agar plated containing a dye hydrolysable by |?-galactosidase. β-Galactosidase-positive colonies are blue; such colonies contain plasmids in which a DNA was successfully inserted. A LIBRARY IS A COLLECTION OF RECOMBINANT CLONES The combination of restriction enzymes and various cloning vectors allows the entire genome of an organism to be packed into a vector. A collection of these different recombinant clones is called a library. A genomic library is prepared from the total DNA of a cell line or tissue. A cDNA library comprises complementary DNA copies of the population of mRNAs in a tissue. Genomic DNA libraries are often prepared by performing partial digestion of total DNA with a restriction enzyme that cuts DNA frequently (e.g., a four base cutter such as TaqI). The idea is to generate rather large fragments so that most genes will be left intact. The BAC, YAC, and Pl vectors are preferred since they can accept very large fragments of DNA and thus offer a better chance of isolating an intact gene on a single DNA fragment. A vector in which the protein coded by the gene introduced by recombinant DNA technology is actually synthesized is known as an expression vector. Such vectors are now commonly used to detect specific cDNA molecules in libraries and to produce proteins by genetic engineering techniques. These vectors are specially constructedto contain very active inducible promoters , proper in-phase translation initiation codons , both transcription and translation termination signals, and appropriate protein processing signals, if needed. Some expression vectors even contain genes that code for protease inhibitors , so that the final yield of product is enhanced. A variety of molecules can be used to "probe" libraries in search of a specific gene or cDNA molecule or to define and quantitate DNA or RNA separated by electrophoresis through various gels. Probes are generally pieces of DNA or RNA labeled with a P-containing nucleotide?aor fluorescently labeled nucleotides (more commonly now). Importantly, neither modification (P or fluorescent-label) affects the hybridization properties of the resulting labeled nucleic acid probes. The probe must recognize a complementarysequence to be effective. A cDNA synthesized from a specific mRNA can be used to screen either a cDNA library for a longer cDNA or a genomic library for a complementary sequencein the coding region of a gene. A popular technique for finding specificgenes entails taking a short amino acid equence and, employing the codon usage for that species, making an oligonucleotide probe that will detect the corresponding DNA fragment in a genomic library. If the sequences match exactly, probes 15-20 nucleotides long will hybridize. cDNA probes are used to detect DNA fragments on Southern blot transfers and to detect and quantitate RNA on Northern transfers and to detect and quantitate RNA on Northern blot transfers. Specific antibodies can also be used as probes provided that the vector used synthesizes protein molecules that are recognized by them.

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