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In this article, we will discuss that gene editing is a kind of genetic engineering which includes the insertion, deletion, or replacement of DNA at particular sites in the genome. Moreover, we will also describe and explain the steps included in the polymerase chain reaction (PCR) to clone and amplify DNA, including the role of Taq polymerase. So, let us get started.
Gene Editing
Gene editing can be defined as:
A technique that enables genetic engineers to modify the DNA of an organism through insertion, deletion, or replacement of DNA at specific sites in the genome which cause disease
It is a kind of genetic engineering which does not involve the introduction of foreign DNA into the genome. Through gene editing, scientists are able to manipulate the genome more accurately. For this purpose, incorrect methods that used vectors were employed in the past. These methods are listed below:
- Altering viruses for the insertion of DNA into the gene that causes the disease. This method led to the insertion of DNA into other genes which resulted in unpredictable consequences
- Liposomes which refer to the tiny spheres of lipid molecules that contain the normal gene are sprayed into the noses. This solution was short-term because the epithelial cell that lines the nasal passageway has a short life
New Gene Editing Techniques
Today, scientists are using novel gene editing techniques, among which the most common is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). In this technique, natural defense mechanism bacteria and some archaea are employed. Using this technique, scientists cut the DNA strands at a specific point which is determined by a guide RNA attached to an enzyme (Cas9). After cutting, scientists can either insert, delete or replace defective DNA with normal DNA.
Applications of CRISPR-Cas9
CRISPR has been used in many ways:
- It was applied in the early embryo to create GMOs (Genetically Modified Organisms)
- Scientists have also injected it into the bloodstream of laboratory animals to obtain gene editing in subsets of tissues
- Techniques based on CRISPR-Cas9 have been employed to alter the genomes of farm animals, crop plants, and laboratory model organisms which include rats, mice, and non-human primates.
- With CRISPR-Cas9 technology, scientists were able to alter the genomes of bacteriophages which helped them to develop techniques to annihilate antibiotic-resistant bacteria
- Although CRISPR-Cas9 is most commonly employed as a research tool, however, it is expected that in the future, this technology will be used to cure genetic disorders
Gene Therapy
Gene editing is used in gene therapies, for instance, to develop treatments for cystic fibrosis and sickle cell anemia. We can define gene therapy as:
Treating a genetic disease by modifying the genotype of a person is referred to as gene therapy
Since scientists are learning more about the human genome from the Human Genome Project and the proteome, and they have the latest technology to process huge amounts of data through computational biology, therefore they are gaining a sound understanding of the types of genes that cause genetic diseases. Besides this, they have also become more aware of the location of these genes. Consequently, they know what base changes are required to cure that genetic disease.
In the next section of the article, we will discuss and explain the steps included in the polymerase chain reaction (PCR) to clone and amplify DNA, including the role of Taq polymerase.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a common molecular biology technique that is employed in the majority of gene technology applications.
The example includes DNA profiling to identify criminals and determine paternity or genetic engineering. We can describe it as in vitro method of DNA amplification which is employed to generate large quantities of specific parts of DNA or RNA from extremely small quantities (even a single molecule of DNA or RNA).
Through PCR, scientists have billions of identical copies of DNA or RNA samples in just a few hours. Three primary stages per cycle are included in the PCR process. DNA doubles in every cycle so that in a standard run of 20 cycles, a million DNA molecules are produced. These three stages take place in a PCR instrument or thermal cycler which automatically provides the ideal temperature for each stage and controls the length of time spent at each stage.
The Three Stages of PCR Process
The three stages of the PCR process are:
- Denaturation: In this stage, the double-stranded DNA is heated to 95o Consequently, hydrogen bonds that bond the two DNA strands together are broken.
- Annealing: In this stage of the PCR process, the temperature is decreased to between 50 - 60°C in order to anneal the primers (forward as well as reverse) to the ends of the single strands of DNA.
- Elongation or Extension: In this stage of the PCR process, the temperature is increased to 72°C for at least one minute. This is done because this is the ideal temperature for Taq polymerase to construct the complementary strands of DNA to produce new identical double-stranded DNA molecules.
Which elements are required for each PCR reaction?
Each PCR reaction needs the following elements:
- Target DNA or RNA that is being magnified
- Primers whether forward or reverse. Primers refer to the short sequences of single-stranded DNA whose base sequences are complementary to the 3’ end of the copied DNA or RNA. They determine the region that should be magnified by signaling the DNA polymerase where to start building the new strands
- DNA polymerase: DNA polymerase refers to an enzyme that is used to build the new DNA or RNA strand. The widely used polymerase is Taq polymerase because it originates from the thermophilic bacterium Thermus aquaticus. It implies that it does not undergo denaturation at a high temperature during the first stage of PCR reaction. Moreover, its ideal temperature is sufficiently higher to prevent toughening of the DNA strands that are not copied yet.
- Free nucleotides: They are employed in building DNA or RNA strands
- Buffer solution: It provides the optimum pH for the reactions to take place
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