You must have heard about genetic engineering in newspapers,TV shows,the internet etc. Sci-fi movies like X-men have also aroused your imaginations as to what miracles are possible just by altering genetic material. It must have raised curiosity as to what exactly is genetic engineering and how does it concern you as an individual.
Genetic engineering involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way.
The terms used to describe genetic engineering are : gene manipulation,Recombinant DNA (rDNA) technology,Gene cloning, Genetic modifications or even New genetics. A genetic engineer is an individual who is involved in genetic manipulations
PCR is known as polymerase chain reaction. It is efficient because it multiplies the DNA exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of DNA that is made can serve as a template for new ones.
2. Restriction Enzymes (Molecular Scissor)
The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments, of varying sizes. These can be separated using gel electrophoresis or chromatography.
Purifying DNA from a cell culture, or cutting it using restriction enzymes wouldn’t be of much use if we couldn’t visualize the DNA – that is, find a way to view whether or not your extract contains anything, or what size fragments you’ve cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts.
4. DNA Ligase
In genetic research it is often necessary to link two or more individual strands of DNA, to create a recombinant strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5′ ends, respectively.
The groups of enzymes that catalyse the synthesis of nucleic acid molecules are collectively referred to as polymerases. It is customary to use the name of the nucleic acid template on which the polymerase acts . The three important polymerases are given below.
a. DNA-dependent DNA polymerase that copies DNA from DNA.
b. RNA-dependent DNA polymerase (reverse transcriptase) that synthesizes DNA from RNA.
c. DNA-dependent RNA polymerase that produces RNA from DNA
6. Escherichia coli- A prokaryotic host
The bacterium, Escherichia coli was the first organism used in the DNA technology experiments and continues to be the host of choice by many workers. Undoubtedly, E.coli, the simplest Gram negative bacterium (a common bacterium of human and animal intestine), has played a key role in the development of present day biotechnology.
Under suitable environment, E.coli can double in number every 20 minutes. Thus, as the bacteria multiply, their plasmids (along with foreign DNA) also multiply to produce millions of copies, referred to as colony or in short clone. The term clone is broadly used to a mass of cells, organisms or genes that are produced by multiplication of a single cell, organism or gene.
7. Eukaryotic Host
Eukaryotic organisms are preferred to produce human proteins since these hosts with complex structure (with distinct organelles) are more suitable to synthesize complex proteins. The most commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae. It is a non-pathogenic organism routinely used in brewing and baking industry. Certain fungi have also been used in gene cloning experiments.
8. Selection of Small Self-Replicating DNA
Small circular pieces of DNA that are not part of a bacterial genome, but are capable of self-replication, are known as plasmids. Plasmids are often used asvectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together generating what is known as a recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, recombinant plasmids containing bacteriophage genes.
9. Method to Move a Vector into a Host Cell
The process of transferring genetic material on a vector such as a plasmid, into new host cells, is called transformation. This technique requires that the host cells are exposed to an environmental change which makes them “competent” or temporarily permeable to the vector. Electroporation is one such technique. The larger the plasmid, the lower the efficiency with which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus or other viral vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but may cause insertion of DNA in parts of our chromosomes where we don’t want it, causing complications and even cancer.
10. Methods to Select Transgenic Organisms
Not all cells will take up DNA during transformation. It is essential that there be a method of detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance and transgenic cells can be selected based on expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.