Recombinant DNA Technology: Tools, Processes and Applications

A piece of DNA which has been made by combining two different species, is called recombinant DNA. This kind of DNA is possible to be made as DNA molecules from all organisms share the same chemical formats and it is different only in the nucleotide sequence. Recombinant DNA molecules are also named as chimeric DNA. 

The technology which is used to produce artificial DNA through the combination of various genetic sources, is called recombinant DNA technology. This Technology is also named as genetic engineering. Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School proposed first the idea of recombinant DNA. 

Keyterms: DNA, Recombinant DNA, Recombinant DNA technology, Genetic engineering, Enzymes, Restriction enzymes, Endonucleases, Exonucleases

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Introduction

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The first publication related to recombinant DNA appeared in 1972 and 1973 from Stanford and UCSF. Paul Berg, a professor at Stanford and an author was awarded the Nobel prize in Chemistry for his work on nucleic acids with the subject of recombinant DNA. In 1978, Werner Arber, Hamilton Smith, Daniel Nathans contributed to the discovery of restriction endonucleases which developed the recombinant DNA technology. 

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Tools of Recombinant DNA technology 

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There are three types of enzymes in recombinant DNA technology. The first is restriction enzymes which help to cut, the second one is polymerases which help to synthesize and the last is ligases which help to bind. The restriction enzymes have a major role in recognising the place where the desired gene is put into the vector genome. There are two types of restriction enzymes - Endonucleases and Exonucleases.

Exonucleases drive away the nucleotides from the ends of the strands but endonucleases cut Within the DNA strand.

Endonucleases cut the DNA at specific points and scan the length of the DNA. This specific site of cutting is called the restriction site. Basically, endonucleases are sequence specific. They usually have palindrome sequences. The restriction site gives a sticky ends in the sequence and desired genes, the vectors are cut by the same restriction enzymes. The enzymes also make the work of the ligases easy by obtaining the complementary sticky notes which help to bind the desired gene to the vector.

The vector plays a very important part in the tools of recombinant DNA technology. They are the carrier and integrator of desired genes into the host organism. Plasmids and bacteriophages are the two most common vectors used in recombinant DNA technology for their high copy number. The vectors are built with an origin of replication. These replications start from a sequence of nucleotides. There are genes which obstruct certain antibiotics like ampicillin and cloning sites. These sites are also recognised by restriction enzymes.

Host organism is an important tool of recombinant DNA technology. The desired DNA with the help of the enzymes and through vectors comes here. There are different ways through which these desired DNAs are put into the host. Biolistics, alternate cooling and heating, use of calcium ions etc are some ways to insert the desired DNA into the host.

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Process of Recombinant DNA Technology 

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The process of recombinant DNA technology goes with some steps to produce the expected product.

Step 1- Isolation of Genetic Material.

It is the primary step of recombinant DNA technology in which the desired DNA is isolated in its pure form. It is free from other macromolecules.

Step 2- Cutting the gene at the recognition sites.

In recognising the location the restriction enzymes take a great role and the desired gene is put into the vector genome at that location. This process is called ' restriction enzyme digestion’

Step 3- Amplifying the gene copies through Polymerase chain reaction ( PCR)

In this process a single copy of DNA is enlarged into thousands to millions of copies and after using the restriction enzymes the proper gene of interest is cut.

Step 4- Ligation of DNA Molecules.

In this step there is the union of two pieces as - a fragment of DNA and the vector with the help of enzyme DNA ligase.

Step 5- Insertion of Recombinant DNA into Host.

The process of insertion of recombinant DNA may be called transformation. In this method, the recombinant DNA is introduced with a host cell. After the insertion of recombinant DNA into the Host cell, it becomes multiplied and is exposed in the manufactured protein form under favourable conditions. The transformed cells bring forward the recombinant gene to the offspring.

Read More: Cloning Vector

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Application of Recombinant DNA Technology 

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• In agricultural fields Recombinant DNA Technology plays a major role. It produces genetically-modified organisms such as flavor save tomatoes, golden rice rich in protein and lot more
• Recombinant DNA technology is also used to produce Insulin.
• ELISA is one kind of clinical diagnosis where recombinant DNA technology is used.
• Recombinant DNA technology prevents hereditary diseases through gene therapy and also detects the presence of HIV in a person.

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DNA Cloning 

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A clone means a bunch of individual cells dismounted from one ancestor.clones are genetically identical because of producing daughter cell everyone. Scientists are able to make multiple copies from a single particle of DNA. The gene constitutes a DNA clone and creates identical copies.

DNA Cloning is a process in which the particle of a DNA enters into a tiny DNA molecule. The tiny replicating molecule is known as the carrier of the DNA Vector.

Some of the most commonly used vectors are viruses, plasmids,yeast cells.plasmids are not a part of the main cellular genome but are DNA molecules which are introduced from bacteria. It carries genes with so many benefits such as mating ability, drug resistance.plasmids are small enough for easy manipulation and can carry extra DNA, weaved into them.

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Applications of Gene Cloning

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There are some fields where the gene cloning process can be applicable. These are referred below - 

• Gene therapy is a process where gene cloning is used. A defective gene is replaced by a healthy gene. Leukaemia and sickle cell anaemia etc are some medical diseases which can be treated with gene therapy.
• In the field of Science, gene cloning can be applied to identify and detect a clone which contains a particular gene. It grows in a controlled environment and manipulates that area.
• Gene cloning process is used in agricultural fields to enhance the productivity of crops. Nitrogen fixation is made by cyanobacteria which enhances the fertility of the soil and at the same time reduces the use of fertilizers.
• Production of hormones, vitamins and antibiotics etc depend on the gene cloning process.

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