What Is Genetic Engineering

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“A molecular technique used to directly manipulate, modify or alter genes or genomes in order to manipulate phenotypes is called genetic engineering.”

In this technique, recombinant DNA is constructed and inserted into the host genome using a vector. Or we can remove some mutated sequences from the genome. The first recombinant DNA was built by Paul Berg in 1972.

What Is Genetic Engineering

What Is Genetic Engineering

It is used for the production of improved plant species, therapeutic drugs or proteins, the prevention of hereditary genetic disorders and the construction of genetically modified organisms.

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Humans have long manipulated the genetic material of many organisms. Using selective breeding and cross-hybridization, we have created economically important plant species.

The purpose of developing genetic engineering or genetic manipulation techniques is to create organisms or phenotypes that are useful to us. Genetic engineering techniques are used for

“In genetic engineering, the DNA of two different cells is joined and inserted into the host genome by a vector.” Important elements of gene manipulation experiments are described here.

Vector: Using plasmid DNA as a vector, the gene of interest is inserted into the host genome. Vectors are types of carriers that carry genetic material.

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“A technique used to insert or remove a mutant gene or manipulate the genome of an organism is called genetic engineering.” History of genetic engineering:

The term genetic engineering was first used by science fiction novelists, not just any scientist. In 1951, Jack Williamson first used the term “genetic engineering” in the novel “Dragon Island”.

Shortly thereafter, Watson and Crick discovered the molecular structure of DNA, although genetic experiments had been known since Mendel’s time.

What Is Genetic Engineering

The first recombinant DNA was built by Paul Berg in 1972. In the same year, Herbert Boyer and Stanley Cohen conducted gene transfer experiments. In 1974, Rudolf Jaenisch created genetically modified mice for the first time in the history of genetics.

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During this period (1960-1990), techniques such as restriction digestion, ligation and PCR were discovered that gave wings to the technique of genetic engineering.

Recombinant DNA technology is a type of genetic engineering technology in which an artificial DNA molecule is constructed by joining two different DNAs by physical means. For this purpose, the gene of interest is inserted into a plasmid vector and used for gene transfer experiments.

Gene editing – A gene editing technique is used for genome editing in which an unwanted DNA sequence is deleted or a new gene can be inserted into the host genome. CRISPR-CAS9, TALEN, and ZFN are some of the popular gene editing tools used in gene therapy experiments.

Genetic engineering is used for different purposes, so we first need to establish the purpose of the experiment. The entire genetic engineering process can be broken down into 5 broad steps:

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The gene must contain the DNA sequence that we want to analyze, and the gene has some special characteristics. The candidate gene should have a high GC content and a lower DNA repeat sequence.

Moreover, the gene of interest should not be too long – only a few kb genes can be successfully inserted. The longer the gene, the greater the chance of failure. The candidate gene must contain both a start codon and a stop codon. Related Article: What is the genetic code?

Now the gene of interest can be isolated from the rest of the DNA by restriction digest or polymerase chain reaction.

What Is Genetic Engineering

Restriction endonuclease enzymes are bacterial enzymes capable of digesting a DNA sequence at a specific site. Using a specific type of restriction endonuclease we can cut out and isolate the gene of interest.

What Is Genetic Engineering?

In the polymerase chain reaction, the gene of interest or candidate gene is amplified during thermocycling using gene sequence information.

The machine uses the polymerase chain reaction to make millions of copies of the gene of interest. The amplified gene is isolated by agarose gel electrophoresis.

If the gene we are interested in is well researched beforehand, information about the gene is available in the genetic library and we can use it to artificially synthesize the gene we are interested in. (By using information from a genetic library, a gene can be artificially synthesized)

In the next step, perform DNA cleaning if necessary. Now our DNA is ready to be inserted into the plasmid. Plasmid selection and construction:

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The selection of a plasmid for a genetic engineering experiment is one of the key steps in the entire experiment. Before selecting a plasmid, we need to understand why the plasmid is used in gene transfer experiments.

Scientists use it as a vehicle to transfer a gene of interest to a target location in the genome. It can efficiently move the gene to its target location. The structure of the plasmid is shown in the figure below,

The plasmid must contain an origin of replication, a promoter region, an antibiotic resistance gene, and other sequences of importance. Using a restriction digest method, the insertion site is inserted into the plasmid to which the gene of interest has been ligated.

What Is Genetic Engineering

Using T4 DNA ligase as a potency sealer, it was inserted and ligated into our plasmid DNA of interest. Along with the plasmid, a selectable marker is also inserted into the plasmid DNA to identify recombinant DNA.

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In addition, the promoter region and terminator sequences are also included in the plasmid for efficient expression of the gene of interest. The plasmid containing the gene of interest and other essential sequences is now called a recombinant DNA molecule.

If we do gene cloning, the plasmid is inserted into the bacterial host, usually E. Coli is used for this purpose. When bacteria start to divide, our recombinant plasmid DNA replicates with it.

We now have multiple copies of our plasmid DNA that are extracted with the plasmid DNA extraction kit and used for transformation experiments.

Transport of recombinant DNA into the recipient cell or host genome is another tedious and difficult task. Different methods of inserting recombinant DNA are used for different cell types because no single method can be applied to all cell types. Different transformation methods:

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Microinjection: a sharp needle is used to insert DNA directly into the nucleus of the cell; however, the method is less efficient and requires a higher level of knowledge.

Electroporation: One of the best methods with a high success rate is the electrophoresis method, in which recombinant DNA is introduced into the host’s genome by the passage of an electric current through the cell.

Another good method is sonication – sonication is sometimes used in gene transfer experiments in which recombinant DNA is introduced into the target cell using ultrasound waves. Ultrasonic waves increase cell permeability.

What Is Genetic Engineering

Liposome-mediated gene transfer – Using an artificial, cell-like outer shell known as liposomes, recombinant DNA can be inserted into the host genome.

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Gene transfer by bacterial infection- This method is one of the popular methods and is common in plant genetic engineering experiments. Here, the plant species is infected with bacteria modified to introduce the gene of interest.

Agrobacterium tumifecian is used to introduce recombinant DNA into a plant cell. The gene of interest is inserted into the Agrobacterium Ti plasmid. Plant cells are infected by this bacterial cell culture, and transformed cells are regenerated by plant tissue culture methods.

Chemistry in gene transfer – metal ions, chemicals and solutions of various chemicals are also used in gene transfer experiments; however, the success rate is too low compared to other methods. Insertion confirmation:

Now we have to decide whether to insert the recombinant DNA into our target cell. For this purpose, various molecular genetics technologies are used. In traditional culture methods, the presence or absence of a selectable marker is used to distinguish transformed cells from non-transformed cells.

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However, it is not required for the PCR-based detection method. The detection method based on the polymerase chain reaction is more reliable than other methods.

DNA is extracted from the transformed cell and amplified using primers that are complementary to the gene of interest or our recombinant DNA.

If recombinant DNA is present it will inevitably be amplified otherwise it will not be amplified. In order to conform the two factors, a set of primers complementary to the specific recombinant DNA and a set complementary to the selectable marker sequence are taken and a multiplex PCR reaction is performed.

What Is Genetic Engineering

What happens if a mutation occurs in the gene of interest during the experiment? Because PCR can only amplify DNA. We need sequence information to detect the mutation.

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DNA is extracted from transformed cells and the gene of interest is amplified by PCR. Now PCR amplicons are used for DNA sequencing, where the sequence of the gene of interest is determined using fluorescence chemistry.

Once all the parameters for determining the gene of interest have been met, our cells are now ready for injection into the host organism or for tissue culture experiments. Applications of genetic engineering:

Genetic engineering has great industrial and agricultural value. It is used in medicine, genetic research, agriculture, cultivation improvement and the production of medicinal drugs.

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