By using the techniques of genetic engineering scientists are able to modify genetic materials so that a particular gene of interest from one cell can be incorporated into a different cell. It is necessary to obtain a gene to modify genetic material. First a scientist isolates plasmid DNA from bacteria and DNA carrying a gene of interest from cells of another organism, such as an animal. A piece of DNA containing the gene is inserted into a plasmid, producing recombinant DNA, and the recombinant plasmid is returned to a bacterial cell. This cell is then grown in culture forming a clone of cells.
The foreign DNA spliced into the plasmid is replicated with the rest of the plasmid as the host cell multiplies. In this way, the gene of interest is cloned. A critical step in gene cloning is the identification of the bacterial clone carrying the gene of interest. Gene cloning and genetics engineering were made possible by the discovery of restriction enzymes. These enzymes protect the bacteria against intruding DNA from other organisms, such as phages or other bacteria cells. They work by cutting up the foreign DNA, a process called restriction.
Most restriction enzymes are very specific, recognizing short nucleotide sequences in DNA molecules and cutting at specific points within these sequences. The bacterial cell protects its own DNA from restriction by adding methyl groups(CH3)to adenines or cytosines within the sequence recognized by the restriction enzyme. The restriction fragments are double-stranded DNA fragments with at least one single-stranded end, called a sticky end. These short extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same enzymes.
The unions formed in this way are only temporary, because only a few hydrogen bonds hold the fragments together. The DNA functions can be made permanent , however, by the enzyme DNA ligase, which seals the Strands by catalyzing the formation of phosphodiesterbonds. We now have recombinant DNA, that has been spliced together from two different sources. There are five basic steps included in modifying genetic material so that a particular gene of interest from one cell can be incorporated into a different cell .
Step 1: Isolation of vector and gene-sources DNA. Step 2:Isolation of vector and gene-source DNA. Step 3: introduction of the cloning vector into cells. Step 4: Cloning of cells and also foreign genes. Step 5 : Identification of cell clones carrying the gene of interest. To determine whether the gene was successfully incorporated we can synthesize a probe complementary to it. We trace the probe, which will hydrogen-bond specifically to complementary single strands of the desired gene ,by labeling it with a radioactive isotope or a fluorescent tag.
An example of how gene transfer and incorporation have been used in biomedical or commercial application is gene therapy of insulin. One of the first practical applications of gene splicing was the production of mammalian hormones and other mammalian regulatory proteins in bacteria. Human insulin and human growth hormone (HGH) were among the earliest examples. This insulin produced in this way has greatly benefited the 2 million diabetics in the United states who depend on insulin treatment to control their disease.