You may have heard about genetic engineering in newspapers, TV shows, and the Internet. Sci-fi movies like X-Men depict individuals with enhanced genetic modification that give them special abilities.
So, what exactly is Genetic Engineering? Genetic engineering involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way.
Here are ten tools that are commonly used in genetic engineering:
PCR is known as the 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, 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.
3. Gel Electrophoresis
Purifying DNA from 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 catalyze 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.
- DNA-dependent DNA polymerase that replicates DNA from DNA.
- RNA-dependent DNA polymerase (reverse transcriptase) that transcribes DNA from RNA.
- DNA-dependent RNA polymerase that transcribes RNA from DNA
6. 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 a suitable environment, the number of E. coli can double every 20 minutes. As the bacteria multiply, their plasmids (along with foreign DNA) also multiply to produce millions of copies, referred to as a colony or in a short clone. The term ‘clone’ broadly refers to a mass of cells, organisms, or genes that results from the multiplication of a single cell, organism, or gene.
7. Eukaryotic Host
Eukaryotic organisms are preferred to produce human proteins since these hosts with complex structures (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 the 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 as vectors 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.
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 the 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. Therefore, it is essential to identify the cells that undergo a transformation and those that have not. Generally, plasmids carry genes for antibiotic resistance, and transgenic cells can be selected based on the 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.