What are the steps of recombinant DNA technology

Recombinant DNA technology

Recombinant DNA technology is a method of joining two diverse molecules of DNA. Here in this article, you will find what are the steps of recombinant DNA technology, its principle, and also its applications. Bacteria is the key to recombinant DNA technology as it is reproduced rapidly.

Principle:

In rDNA technology, the desired DNA is isolated in its pure form that means it is free from other macromolecules. Restriction enzymes work like molecular scissors and cut the DNA at specific sites. This is known as restriction enzyme digestion. Purified DNA and interest vector are cut with the same restriction enzyme which provide DNA fragment and vector that is now open. These two pieces are joined together by the DNA ligase enzyme. The resulting molecule of DNA is a mixture of two DNA molecules. Through PCR multiple DNA sequence copies can be produced in in-vitro by DNA polymerase. It helps the sequence to amplify into a single copy or few DNA copies into thousands to millions of copies.

Material required:

• Plasmid
• Endonucleases
• Exonucleases
• DNA Ligase
• DNA template
• Thermal cycler
• Primer
• Master mix
• Pcr tubes

Steps involved in the procedure:

Isolation of genetic material

Firstly, the desired DNA of rDNA technology is isolated in its pure form that means it is free from other macromolecules.

Since, DNA found within the cell membrane besides other molecules like RNA, polysaccharides, proteins, and lipids it must be separated and then purified. It must be separated from enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases, etc. Other macromolecules are deletable with other enzymes or treatments. Ultimately, ethanol addition causes DNA to precipitate out as fine threads. Finally, This is coiled out to give purified DNA.

Restriction enzyme digestion

Secondly, there is a step of restriction enzyme digestion. Restriction enzymes work like molecular scissors and cut the DNA at specific sites. This is known as restriction enzyme digestion. They have incubation for purified DNA with selected restrictions enzyme at an optimal situation for that special enzyme.

The technique agarose gel electrophoresis reveals the restriction enzyme digestion progression.

This technique included running out DNA on an agarose gel.

Vector DNA also processed by using the same process.

Amplification by using PCR

Through PCR multiple DNA sequence copies can be produced in in-vitro by DNA polymerase. With the help of PCR, we can amplify sequences into single copies or few DNA copies into thousands to millions of copies.

PCR reactions are runs on thermal cyclers by using the following components

• Template-DNA to be amplified
• Primers-small, chemically produced oligonucleotides and complementary to the DNA region.
• Enzyme-DNA Polymerase
• Nucleotides-needed to expend primers by the enzyme.
• The cut DNA fragments can be amplified by using PCR and then ligated with a cut vector.

Ligation of DNA molecules

Purified DNA and vector of interest are cut with the same restriction enzyme. This in turn gives cut DNA fragment and vector that is now open.

These two pieces are joined together by the DNA ligase enzyme. The resulting molecule of DNA is a mixture of two DNA molecules. In genetic terminology intermixing of different DNA strands is called recombinant.

So, a new hybrid DNA molecule is called recombinant DNA molecule, and technology referred to as recombinant DNA technology.

Insertion of recombinant DNA into the host

Specifically, recombinant DNA is entered into a recipient cell of the host. This process is known as “ Transformation ”.

Foreign DNA can not be easily acceptable by bacterial cells. So, they are treated to make able them “competent” for new DNA acceptance.

Thermal shock, Ca⁺² ion treatment, and electroporation, etc.

Isolation of Recombinant cells

Due to the transformation process, a mixed population of transformed and non-transformed host cells is formed. In the selected process only transformed host cells are filtered. The marker gene of the plasmid vector is used for the isolation of recombinant cells from the non-recombinant cells. For example, the PBR322 plasmid vector involves various marker genes when pst1 RE is utilized it can knock out an ampicillin-resistant gene from the plasmid so, recombinant cells become sensitive to ampicillin.

Result and its interpretation:

Consequently, desirable traits and qualities can be produced by these methods. Here, Isolated DNA can be added to any organism of our interest and we can get many biological molecules.

Applications of recombinant DNA technology:

• Recombinant DNA is extensively used in bioengineering as well as in medicine, and research.
• Recombinant DNA technology is applicable in the identification as well as in mapping, gene sequencing, and determination of their function.
• Similarly, many other practical applications of rDNA are found in industry, food production, human, veterinary sciences, agriculture, and bio-engineering.
• Likewise, the presence of HIV in a person can be detected by rDNA technology.
• Further, recombinant DNA technology is valuable in agriculture for the manufacturing of Bt-cotton to shelter plants against ball worms.
• Finally, Human insulin can be produced by rDNA technology.

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