Recombinant DNA Technology

The exchange of genetic information between DNA segments of the same species is termed genetic recombination. Nonetheless, with the advancement of technology, one can transfer genes of one species to another artificially.

In this article, you will find fundamental details of the artificial method - recombinant DNA technology and gain insight into the steps involved accordingly.

The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.

It is the technology to produce an artificial DNA molecule by combining two or more fragments of DNA that are not necessarily associated with each other. Usually, such DNA fragments are obtained from several biological sources.  

Recombinant DNA is DNA from two distinct species injected into a host organism to create new genetic combinations useful in science, medicine, agriculture, and industry. Laboratory geneticists' primary purpose is to identify, define, and modify genes, as the gene is the focus of all genetics. Consider that each human cell has about 2 meters (6 feet) of DNA. As a result, even a small piece of tissue can contain thousands of kilometers of DNA. Recombinant DNA technology, on the other hand, has made it possible to isolate a single gene or other section of DNA, allowing researchers to establish its nucleotide sequence, investigate its transcripts, alter it in extremely specific ways, and reintroduce the transformed sequence into a living creature.

Notably, several steps are followed to recombine DNA segments. Furthermore, under an ideal situation, a recombinant DNA molecule can replicate by entering a cell.

The said technology is also known as genetic engineering. In a broader sense, it is created through three different methods – transformation, non-bacterial transformation, and phage introduction. 

Tools of rDNA Technology

Recombinant DNA Technology's Implementation Tools are as follows:

Restriction enzymes, for example, aid in cutting, polymerases aid in synthesising, and ligases aid in binding. In recombinant DNA technology, restriction enzymes play an important role in deciding where the desired gene is inserted into the vector genome. Endonucleases and Exonucleases are the two types of endonucleases.

Exonucleases remove the nucleotides off the ends of the strands, whereas Endonucleases cut within the DNA strand. Restriction endonucleases are sequence-specific enzymes that cleave DNA at specified locations and are frequently palindrome sequences. They examine the length of DNA and cut it at a specified location known as the restriction site. As a result, the sequence has sticky ends. The complimentary sticky notes are obtained by cutting the desired genes and vectors with the same restriction enzymes, enabling the work of the ligases to bind the desired gene to the vector much easier.

The vectors aid in the transport and integration of the desired gene. These are the ultimate vehicles that transfer the desired gene into the host organism, hence they are a crucial part of the recombinant DNA technology toolkit. Because of their large copy number, plasmids and bacteriophages are the most commonly employed vectors in recombinant DNA technology. The vectors are made up of an origin of replication, which is a nucleotide sequence from which replication begins, a selectable marker, which are genes that show antibiotic resistance, such as ampicillin resistance, and cloning sites, which are sites recognised by restriction enzymes where desired DNAs are inserted.

The recombinant DNA is injected into the host organism. The host is the most powerful instrument in recombinant DNA technology, as it uses enzymes to take in the vector that has been modified with the desired DNA.

These recombinant DNAs are injected into the host in a variety of ways, including microinjection, biolistics or gene gun, alternate cooling and heating, calcium ion usage, and soon.

Goals of rDNA Technology

Some of the goals of this technology  are mentioned below: 

• Isolation and characterization of genes.

• Desired modification in isolated genes.

• Artificial synthesis of new genes.

• Modification of organisms’ genome.

• Interpretation of hereditary diseases and related cures.

• Enhancement of the human genome.

Steps of Recombinant DNA Technology

1. DNA Isolation 

• DNA is isolated in its pure form, which means they are devoid of other macromolecules.

• In rDNA technology, the initial step is to extract the desired DNA in its purest form, that is, free of extraneous macromolecules.

• Because DNA coexists with other macromolecules such as RNA, polysaccharides, proteins, and lipids within the cell membrane, it must be separated and purified using enzymes such as lysozymes, cellulase, chitinase, ribonuclease, and proteases.

• Other enzymes or treatments can remove other macromolecules. The DNA eventually precipitates out as fine threads as a result of the presence of ethanol. After that, the pure DNA is spooled out.

2. Cutting of DNA/Restriction Enzyme Digestion

• For this step, the restriction enzymes are quite vital. It helps to identify the location wherein a designated gene is introduced into a vector genome. The said reaction is known as restriction enzyme digestions. 

• They entail incubating pure DNA with a restriction enzyme of choice at conditions that are appropriate for that enzyme.

• The 'Agarose Gel Electrophoresis' technology displays the restriction enzyme digestion's progress.

• This method entails passing the DNA across an agarose gel. When current is applied, negatively charged DNA flows to the positive electrode and is divided into different sizes. This permits the digested DNA fragments to be separated and snipped out.

• The same method is used to process the vector DNA.

3. Amplifying of DNA

• Copies of genes are amplified through PCR or polymerase chain reaction. It is essentially a process to increase a single DNA copy into several copies after the desired gene of interest is cut with restriction enzymes. 

• It allows a single copy or a few copies of DNA to be amplified into thousands or millions of copies.

• The following components are used in PCR reactions that are conducted on 'thermal cyclers':

1. Template: DNA that has to be amplified.

2. Primers: oligonucleotides are tiny, chemically produced oligohnucleotides that are complementary to a DNA region.

3. Enzyme: DNA polymerase.

4. Nucleotides: The enzyme is required to lengthen the primers.

• PCR can be used to amplify the cut DNA fragments, which can subsequently be ligated with the cut vector.

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4. Joining DNA

• The vector and a section of DNA are joined in this step. It is achieved with the help of the enzyme DNA ligase. 

• With the same restriction enzyme, the pure DNA and the vector of interest are cut.

• This yields the cut DNA fragment and the cut vector, both of which are now open.

• Ligation is the process of putting these two parts together with the enzyme 'DNA ligase.'

• The resulting DNA molecule is a hybrid of the interest molecule and the vector DNA molecules. Recombination is the term used in genetics to describe the merging of different DNA strands.

• As a result, this new hybrid DNA molecule is known as a recombinant DNA molecule, and the process is known as recombinant DNA technology.

5. Insertion of rDNA into a Host

• Here rDNA is added to the recipient host cell, and the entire process is called transformation. Post insertion, the recombinant DNA multiplies and manifests as manufactured protein under favorable conditions.

• The recombinant DNA is then transferred into a recipient host cell, most commonly a bacterial cell, in this stage. The term for this procedure is 'Transformation.'

• Bacterial cells have a hard time accepting foreign DNA. As a result, they are given treatments to make them 'capable' of accepting new DNA. Thermal shock, Ca++ ion therapy, electroporation, and other procedures may be applied.

6. Recombinant Cell Isolation

• A mixed population of converted and non-transformed host cells results from the transformation process.

• Only the transformed host cells are filtered during the selection procedure.

• The marker gene of the plasmid vector is used to distinguish recombinant cells from non-recombinant cells.

• PBR322 plasmid vector, for example, comprises two marker genes (Ampicillin resistant gene and Tetracycline resistant gene). When pst1 RE is utilised, it eliminates the Ampicillin resistance gene from the plasmid, causing the recombinant cell to become Ampicillin sensitive.

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Application of Recombinant DNA Technology

Recombinant DNA technology has been widely used in medical science, industries, animal husbandry, and agriculture.

The following highlights the application of r DNA technology in brief -

• To produce recombinant HB vaccines.

• For producing human insulin.

• To facilitate better crop production.

• For producing growth hormones in humans to treat dwarfism.

• For better gene therapy. 

• To acquire DNA fingerprinting.

• To diagnose several types of diseases. 

What is DNA Cloning?

Typically, a clone is defined as a cluster of individual cells that come from a progenitor. A clone is genetically similar to its parent cell from which it replicates. 

DNA cloning is initiated when DNA fragments are inserted into DNA molecules. The said replicating molecule is the carrier of DNA vectors. A clone is a group of individuals or cells that derive from a single progenitor. Clones are genetically identical because each time a cell replicates, it produces identical daughter cells. Scientists have discovered a way to make many copies of a single DNA fragment, a gene that can be used to make identical copies of a DNA clone. DNA cloning is accomplished by inserting DNA pieces into a small DNA molecule. This molecule is designed to multiply inside a living cell, such as a bacteria. The carrier of the DNA vector is a small replicating molecule. Plasmids, yeast cells, and viruses are among the prominent vectors of rDNA technology examples. Plasmids are circular DNA molecules that bacteria inject into the human body. They aren't a part of the cell's core genome. It carries genes that confer favourable traits on the host cell, such as the ability to mate and drug resistance. They can be easily controlled since they are small enough and can carry extra DNA that has been woven into them.

Importance of DNA Cloning:

• Agriculture is one of the fields where gene cloning is used. Nitrogen fixation is carried out by cyanobacteria, and desired genes can be used to boost crop output and improve health. As a result of this method, the consumption of fertilisers is reduced, and chemical-free products are produced.

• In the world of medicine, gene cloning is extremely significant. Hormones, vitamins, and medicines are all made with it.

• It can be used to identify and detect a clone that contains a specific gene that can be modified by growing in a controlled environment. 

• It is utilised in gene therapy, in which a damaged gene is replaced with a healthy one. This technique can be used to cure diseases like leukaemia and sickle cell anaemia.