Recombinant DNA technology| Steps and Applications of Recombinant DNA technology

 Steps of Recombinant DNA Technology

Recombinant DNA technology is an advanced technique of combining two DNA molecules from different species to create a new species.  Here, I will discuss the steps of recombinant DNA technology and the applications of recombinant DNA technology. Recombinant DNA technology has vast applications in health, science, agriculture, and industrial fields.

Recombinant DNA technology

 Recombinant DNA technology is an advanced technique of combining two DNA molecules from different species to create novel species that have applications in many fields including science, health, agriculture, and industry.

Steps of Recombinant DNA technology:

There are steps involved to produce recombinant DNA molecules:

DNA Isolation: 

The first step is to separate DNA molecule from other large molecules and isolate it in their pure form. The DNA must be separated from the cell wall, cell membrane, and other carbohydrates and proteins. This process is called cell lysis.  For this purpose, we need enzymes for breaking macromolecules. Several methods are used for the extraction of genomic DNA such as organic, proteinase K method, and non-organic techniques.  

Restriction Enzymes Cleavage:

Restriction digestion enzymes such as nucleases, and polymerases will cleave the DNA molecule at specific sites.  These molecular scissors cut the double-stranded DNA at the cleavage site producing single-strand DNA. There are two kinds of restriction enzymes: Endonucleases and Exonucleases. When DNA molecule is digested by enzymes, it produces both blunt and sticky ends. This process is done through agarose gel electrophoresis. After extraction of DNA by extraction methods, the DNA is passed through gel electrophoresis. When the current is passed on agarose gel the negatively charged phosphate molecule on DNA binds to the positive electrode. The fragments are then separated based on their size. In this way, vector DNA is cleaved into single-stranded fragments.

Amplification of Genomic DNA:

DNA is amplified by using a Polymerase chain reaction (PCR) that makes several copies of DNA.  First, we take the template DNA and denaturation is done by breaking in single strands, then primers are required for annealing. Primers are short oligonucleotides that are complementary in nature.  These primers bind to the target DNA (single-strand DNA). Then an enzyme called DNA polymerase synthesizes DNA. After synthesis, an extension of template DNA is carried out.  Nucleotides are added to extend the length of the primer.  In this way, DNA segments have been amplified by using PCR and then the joining of DNA molecules is required.

DNA molecule attachment:

When the enzyme breaks down vector DNA and DNA of interest, now we have two separate fragments: one of DNA vector and the other DNA of interest. So we need to join the two fragments for this purpose we perform ligation which is done by using a DNA ligase enzyme. DNA ligation is of two types blunt end ligation and sticky end ligation. DNA ligase joins the overhangs in the DNA strand.  The vector DNA and DNA of interest (plasmid DNA) are joined together making a hybrid DNA molecule and this is known as a recombinant DNA molecule. A hybrid DNA molecule is a mixture of two DNA molecules.

Adding Recombinant DNA to host cell:

The recombinant DNA is inserted into the host cell of a bacterium. This is also called transformation. Foreign DNA is not immediately taken by bacterial cells. The DNA must be treated before insertion into the host cell. The treatment process includes electroporation, heat shock, transduction, and microinjection.

Recombinant DNA molecule isolation:

Now the recombinant DNA is isolated from the host cell. Only the host cells which have entered the bacterium cell are filtered through the selection procedure. Plasmid vectors allow the separation of recombinant and non-recombinant DNA molecules. Different plasmids are used such as PUC, and PBR322. If bacteria E.coli receive recombinant DNA which contains an ampicillin resistance gene then cells develop resistance against ampicillin. PBR322 plasmid provides resistance against both ampicillin and tetracycline genes. For the first time, the recombinant DNA was isolated by E.coli bacterium in 1973.

 Applications of Recombinant DNA Technology

Applications of Recombinant DNA Technology

Applications of recombinant DNA technology have played an important role in medical research.  Functions of genes are identified, mapped, and sequenced through recombinant DNA molecules.  Recombinant proteins are used in biotechnological research labs to develop antibodies for the synthesis of protein in living beings. Recombinant DNA technology has many uses in medicine, pharmaceuticals, industrial fields, the agricultural sector, food production, and the engineering sector.

Medical Applications:

Insulin is manufactured by using recombinant DNA.  The human gene which produces insulin has been introduced in E.coli bacterium to produce insulin at a massive scale. The pancreas of pigs and cows can produce the hormone insulin, which is for diabetes patients. HIV can be identified in a person by recombinant DNA. ELISA used in clinical diagnostic laboratories is also an illustration of recombinant DNA.  Penicillin and Streptomycin antibiotics for fungal strains are also an application of recombinant DNA technology.

Gene Therapy:

Gene therapy is used to fix gene abnormalities that cause hereditary diseases. With the use of recombinant DNA technology, medical laboratories will be able to remove defective genes from disease-causing people that may cause inherited disorders in persons such as hemophilia. The defective gene is converted to a normal gene. 

Vaccine Production: 

 Vaccines are produced that transfer genes coding antigens to microorganisms that cause disease. These antibodies provide a defense mechanism against bacterial or viral infection.

Interferon Production:

 Viruses infected with cells produce interferon, which are proteins in nature. Human blood cells are used to create interferon which provides defense against various kinds of cancers like breast cancer. This is an expensive technique but interferon can be made at less cost by recombinant DNA technology.

Enzyme Production: 

 Recombinant DNA can generate valuable enzymes. For example, the bot clot-dissolving enzyme urokinase enzyme can be able to break down enzymes.

Creation of Transgenic Plants:

 Transgenic Plants are produced by transferring genes from one specie to another through genetic engineering.  Now, these plants have foreign genes. These pants when they go through gene modification produce plants that are more resistant to diseases, pests, and insects. Recombinant DNA technology in the agriculture field results in the production of Bt cotton to defend plants from worms.

Root Nodule development in crop:

  Legume plants produce nitrogen-fixing bacteria in their root bacterium. These bacteria transfer free nitrogen into the air to produce nitrates. By genetic engineering, bacterial genes share free nitrogen with cereal crops.

C4 Plant Production: 

By increasing agricultural plants’ photosynthetic capacity, production will be increased. During the Calvin cycle, when C3 pant is converted to C4 through protoplast fusion or recombinant DNA technology, it enhances the photosynthetic rate.  C3 and C4 plants produce more biomass production.