Genetic Engineering in Medicine

Genetic engineering is the genetic make-up of an organism’s genome using biotechnology tools and the one of the most powerful and promising application of the genetic engineering involves the treatment of genetic disorders like sickle cell anemia, Duchenne muscular dystrophy, cystis fibrosis, Tay-Sachs disease, Huntington’s chorea and Lesch-Nyhan syndrome. Now, medical Scientists can identify more than 3000 disorders happens because of the error in individuals DNA. By this technique scientists modify the genome of an organism. Creation of genetically modified organisms requires recombinant DNA. Recombinant DNA is a combination of DNA from different organisms or different locations in a given genome that would not normally be found in nature. The goal is to add one or more new traits that are not already found in that organism. Examples of genetically engineered organisms currently on the market include plants with resistance to some insects, plants that can tolerate herbicides, and crops with modified oil content.

Steps involved in Genetic Engineering

Choice of host organism and cloning vector

Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.

Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:

• DNA replication origin is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism.
• One or more unique restriction endonuclease recognition sites to serves as sites where foreign DNA may be introduced.
• A selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences.
• A tag gene that can be used to screen for cells containing the foreign DNA.

Preparation of vector DNA

The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see DNA end). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.

Preparation of DNA to be cloned

For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from extinct animals), as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography). Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning.

Creation of recombinant DNA with DNA ligase

The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.

Introduction of recombinant DNA into host organism

Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

Selection of organisms containing vector sequences

Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.

Screening for clones with desired DNA inserts and biological properties

The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations, antibody probes, polymerase chain reaction, restriction fragment analysis and/or DNA sequencing.