How was able to make a recombinant DNA?

Making Recombinant DNA

How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:

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Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated like chemical reagents. Figure 10-1 on the following page gives a general outline of the approach.

The term recombinant DNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and prokaryotes. Recombinant DNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer the alternative name chimeric DNA, after the mythological Greek monster Chimera. Down through the ages, the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different animals. Likewise, recombinant DNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinant DNA technology.
Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 10-2 on page 303. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.top link
Cutting DNA

The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation. Any DNA molecule, from viruses to humans, contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor would they be cut in vivo, because most organisms do not have restriction enzymes.

Let's look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the following sixnucleotide-pair sequence in the DNA of any organism:

more detail : http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mga.section.1549