【文章內(nèi)容簡(jiǎn)介】 , and the linear molecule formed has two sticky ends. Production of these sticky ends is another feature of restriction enzymes that makes them suitable for rebinant DNA technology. The principle is simply that, if two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the same plementary sticky ends, making it possible for DNA chimeras to form. Hence, if both vector DNA and donor DNA are cut with EcoRI, the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed.MESSAGERestriction enzymes have two properties useful in rebinant DNA technology. First, they cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes make staggered cuts that create singlestranded sticky ends conducive to the formation of rebinant DNA.Dozens of restriction enzymes with different sequence specificities have now been identified, some of which are shown in Table 121. You will notice that all the target sequences are palindromes, but, like EcoRI, some enzymes make staggered cuts, whereas others make flush cuts. Even flush cuts, which lack sticky ends, can be used for making rebinant DNA.DNA can also be cut by mechanical shearing. For example, agitating DNA in a blender will break up the long chromosomesized molecules into flushended clonable segments.Joining DNAMost monly, both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form rebinant molecules. Figure 124a shows a plasmid vector that carries a single EcoRI restriction site。 so digestion with the restriction enzyme EcoRI converts the circular DNA into a linear molecule with sticky ends. Donor DNA from any other source (say, Drosophila) also is treated with the EcoRI enzyme to produce a population of fragments carrying the same sticky ends. When the two populations are mixed, DNA fragments from the two sources can unite, because double helices form between their sticky ends. There are many openedup vector molecules in the solution, and many different EcoRI fragments of donor DNA. Therefore a diverse array of vectors carrying different donor inserts will be produced. At this stage, although sticky ends have united to generate a population of chimeric molecules, the sugarphosphate backbones are still not plete at two positions at each junction. However, the backbones can be sealed by the addition of the enzyme DNA ligase, which create phosphodiester bonds at the junctions (Figure 124b). Certain ligases are even capable of joining DNA fragments with bluntcut ends.Amplifying rebinant DNAThe ligated rebinant DNA enters a bacterial cell by transformation. After it is in the host cell, the plasmid vector is able to replicate because plasmids normally have a replication origin. However, now that the donor DNA insert is part of the vector39。s length, the donor DNA is automatically replicated along with the vector. Each rebinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles of cell division will take place, and the rebinant vectors will undergo more rounds of replication. The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified copies of the single donor DNA fragment is the DNA clone (Figure 125).Cloning a specific geneThe foregoing descriptions are generic approaches to creating rebinant DNA. However, a geneticist is interested in isolating and characterizing some particular gene of interest, so the procedures must be tailored to isolate a specific rebinant DNA clone that will contain that particular gene. The details of the process differ from organism to organism and from gene to gene. An important initial factor is the choice of an appropriate vector for the job at hand.Choosing a cloning vectorThe ideal vector is a small molecule, facilitating manipulation. It must be capable of prolific replication in a living cell, thereby enabling the amplification of the inserted donor fragment. Another important requirement is to have convenient restriction sites that can be used for insertion of the DNA to be cloned. Unique sites are most useful because then the insert can be targeted to one site in the vector. It is also important to have a method for easily identifying and recovering the rebinant molecule. Numerous cloning vectors are in current use, and the choice between them often depends on the size of the DNA segment that needs to be cloned and on the intended application for the cloned gene. We shall consider several monly used types.Plasmids.As described earlier, bacterial plasmids are small circular DNA molecules that are not only distinct from the main bacterial chromosome, but also additional to it. They replicate their DNA independently of the bacterial chromosome. Many different types of plasmids have been found in bacteria. The distribution of any one plasmid within a species is generally sporadic。 some cells have the plasmid, whereas others do not. In Chapter 7, we encountered the F plasmid, which confers certain types of conjugative behavior to cells of E. coli. The F plasmid can be used as a vector for carrying large donor DNA inserts, as we shall see in Chapter 14. However, the plasmids that are routinely used as vectors are those that carry genes for drug resistance. The drugresistance genes are useful because the drugresistant phenotype can be used to select not only for cells transformed by plasmids, but also for vectors containing rebinant DNA. 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.Two plasmid vectors that have been extensively used in genetics are shown in Figure 126. These vectors are derived from natural plasmids, b