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Attaching donor and vector DNA Most commonly, both donor and vector DNA are digested by a restriction enzyme that produces complementary sticky ends and are then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. Figure 11-5a shows a bacterial plasmid DNA that carries a single EcoRI restriction site; so digestion with the restriction enzyme EcoRI converts the circular DNA into a single linear molecule with sticky ends. Donor DNA from any other source, such as human DNA, also is treated with the EcoRI enzyme to produce a population of fragments carrying the same sticky ends. When the two populations are mixed under the proper physiological conditions, DNA fragments from the two sources can hybridize, because double helices form between their sticky ends (Figure 11-5b). There are many opened-up plasmid molecules in the solution, as well as many different EcoRI fragments of donor DNA. Therefore a diverse array of plasmids recombined with different donor fragments will be produced. At this stage, the hybridized molecules do not have covalently joined sugar–phosphate backbones. However, the backbones can be sealed by the addition of the enzyme DNA ligase, which creates phosphodiester linkages at the junctions (Figure 11-5c ). cDNA can be joined to the vector using ligase alone, or short sticky ends can be added to each end of a plasmid and vector. Another consideration at this stage is that, if the cloned gene is to be transcribed and translated in the bacterial host, it must be inserted next to bacterial regulatory sequences. Hence, to be able to produce human insulin in bacterial cells, the gene must be adjacent to the correct bacterial regulatory sequences. Amplification inside a bacterial cell Amplification takes advantage of prokaryotic genetic processes, including those of bacterial transformation, plasmid replication, and bacteriophage growth, all discussed in Chapter 5. Figure 11-6 illustrates the cloning of a donor DNA segment. A single recombinant vector enters a bacterial cell and is amplified by the replication that takes place in cell division. 

There are generally many copies of each vector in each bacterial cell. Hence, after amplification, a colony of bacteria will typically contain billions of copies of the single donor DNA insert fused to its accessory chromosome. This set of amplified copies of the single donor DNA fragment within the cloning vector is the recombinant DNA clone. The replication of recombinant molecules exploits the normal mechanisms that the bacterial cell uses to replicate chromosomal DNA. One basic requirement is the presence of an origin of DNA replication (as described in Chapter 7). CHOICE OF CLONING VECTORS Vectors must be small molecules for convenient manipulation. They must be capable of prolific replication in a living cell to amplify the inserted donor fragment. They must also have convenient restriction sites at which the DNA to be cloned may be inserted. Ideally, the restriction site should be present only once in the vector because then restriction fragments of donor DNA will be inserted only at that one location in the vector. It is also important that there be a way to identify and recover the recombinant molecule quickly. Numerous cloning vectors are in current use, suitable for different sizes of DNA insert or for different uses of the clone. Some general classes of cloning vectors follow. Plasmid vectors As described earlier, bacterial plasmids are small circular DNA molecules that replicate their DNA independently of the bacterial chromosome. The plasmids that are routinely used as vectors are those that carry genes for drug resistance. These drug-resistance genes provide a convenient way to select cells transformed by plasmids: those cells still alive after exposure to the drug must carry the plasmid vectors containing the DNA insert, as shown at the left in Figure 11-7. Plasmids are also an efficient means of amplifying cloned DNA because there are many copies per cell, as many as several hundred for some plasmids. Examples of some specific plasmid vectors are shown in Figure 11-7. 

Bacteriophage vectors Different classes of bacteriophage vectors can carry different sizes of donor DNA insert. A given bacteriophage can harbor a standard amount of DNA as an insert “packaged” inside the phage particle. Bacteriophage  (lambda) is an effective cloning vector for double-stranded DNA inserts as long as about 15 kb. Lambda phage heads can package DNA molecules no larger than about 50 kb in length (the size of a normal chromosome). The central part of the phage genome is not required for replication or packaging of DNA molecules in E. coli and so can be cut out by using restriction enzymes and discarded. The deleted central part is then replaced by inserts of donor DNA. An insert will be from 10 to 15 kb in length because this size insert brings the total chromosome size back to its normal 50 kb (Figure 11-8). As Figure 11-8 shows, the recombinant molecules can be directly packaged into phage heads in vitro and then introduced into the bacterium. Alternatively, the recombined molecules can be transformed directly into E. Coli. 

In either case, the presence of a phage plaque on the bacterial lawn automatically signals the presence of recombinant phage bearing an insert. Vectors for larger DNA inserts The standard plasmid and phage vectors just described can accept donor DNA of sizes as large as 25 to 30 kb. However, many experiments require inserts well over this upper limit. To meet these needs, the following special vectors have been engineered. In each case, after the DNAs have been delivered into the bacterium, they replicate as large plasmids. Cosmids are vectors that can carry 35- to 45-kb inserts. They are engineered hybrids of phage DNA and bacterial plasmid DNA. Cosmids are inserted into phage particles, which act as the “syringes” that introduce these big pieces of recombinant DNA into recipient E. coli cells. The plasmid component of the cosmid provides sequences necessary for the cosmid’s replication. Once in the cell, these hybrids form circular molecules that replicate extrachromosomal in the same manner as plasmids do.  PAC (P1 artificial chromosome) vectors deliver DNA by a similar system but can accept inserts ranging from 80 to 100 kb. In this case, the vector is a derivative of bacteriophage P1, a type that naturally has a larger genome than that of BAC (bacterial artificial chromosome) vectors, derived from the F plasmid, and can carry inserts ranging from 150 to 300 kb (Figure 11-9). The DNA to be cloned is inserted into the plasmid, and this large circular recombinant DNA is introduced into the bacterium by a special type of transformation. BACs are the “workhorse” vectors for the extensive cloning required by large-scale genome-sequencing projects (discussed in Chapter 12). Finally, inserts larger than 300 kb require a eukaryotic vector system called YACs (yeast artificial chromosomes, described later in the chapter). For cloning the gene for human insulin, a plasmid host was selected to carry the relatively short cDNA inserts of approximately 450 bp.

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