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Synthesis of Recombinant DNA, Its function and Importance
#1
DNA or deoxyribonucleic acid is the hereditary material in humans as well as plants and animals. Every cell in the body has the same structure and function of DNA. It is found in the cell’s nucleus. Mitochondria are cell organelles which contain the small amount of DNA. There are four nucleotide bases which make the structure of DNA that are adenine, guanine, cytosine and thymine. These bases are actually the genetic information stored in the DNA molecules in the form of codes. There are almost 3 billion bases and all the people have 99% similarity with each other in their bases.

Recombinant DNA
Following are three methods through which recombinant DNA is made.

Transformation

- First the piece of DNA is selected which is to be inserted into the vector or plasmid.
- Isolated DNA piece is cut by using restriction enzymes.
- The cut piece is inserted into the vector where it attaches to the genetic material already present in the vector with the enzyme called DNA ligase .
- A genetic marker is used for the identification of recombinant DNA. Usually an antibiotic marker is used.
- This process of inserting vector into the host cell is called as transformation.

During the process of transformation, usually E. coli acts as a host cell. When a foreign gene is inserted into this vector, it is prepared before the insertion so that it accepts the gene. To keep the foreign gene or DNA different from the host cell’s genome, markers, color changes or some other characteristics are used.

Non-bacterial Transformation
This process of synthesis of recombinant DNA is different from the transformation process in the sense that it does not use bacteria as a host for the foreign DNA but other sources are used. For example, DNA microinjection is used in which cell is inserted into the nucleus of the recipient cell directly. Bioballistic is another method in which silver particles are used with the help of gene gun to bombard the recombinant DNA into the recipient cell.

Phage
In this process, a vector is used instead of bacteria as host cell. Phage is a type of virus which acts as a host cell for carrying recombinant DNA. Scientists inactivate the function of virus itself so that when it enters the body, it only inserts the recombinant DNA and does not cause any infections.


Function of Recombinant DNA

When the recombinant DNA enters the host cell, the host cell starts expressing the proteins present in the rDNA. If the expression factors are added along with the rDNA then host cell will be able to produce significant amount of proteins. Expression of the protein will not appear until there are some signals in the host cells. There are specific signals for every species of bacteria for example; E. coli does not get the signals of human terminators and promoters.

Importance of Recombinant DNA
Recombinant DNA is very helpful in techniques like gene therapy. It is helpful in curing different diseases like cancer. Healthy genes are inserted in the body and they replace the defected genes. Some other important features of recombinant DNA are that it can give better yield of crops. Disease like sickle cell anemia and hemophilia can be treated with recombinant DNA because it produces clotting factors. Scientists have successfully produced insulin with the help of recombinant DNA. Pharmaceutical industry has taken advantage of rDNA by making drugs. Recombinant DNA has enabled plants to make their own insecticides.
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#2
The recombinant DNA (rDNA) technology initially appeared during the 1960- 1970s, even though the fundamental principle of recombination was revealed much earlier. In fact, in the year 1928, an English bacteriologist named Frederick Griffith who was researching about the bacteria responsible for an epidemic of pneumonia in London, first illustrated the genetic transformation. In his experiment, the living cells took up genetic material released by other cells and were transformed phenotypically by the new genetic information. More than ten years later, Oswald Avery recapitulated Griffith's experiment in which he isolated the transforming molecule later came to be known as DNA. These early investigations revealed that in the laboratory, DNA can be transferred from one cell to another and thus altering the original genetic phenotype of a life form.

The tradition of cutting, pasting, and copying the DNA can be traced back to Arthur Kornberg's successful replication of viral DNA (DNA polymerase). This was followed by the Werner Arber's breakthrough discovery of restriction enzymes in bacteria that degrade foreign viral DNA while protecting their own DNA. The discovery that ligase enzyme could be utilized to glue the DNA molecules together accelerated the rDNA technology research, popularly known as playing god in the history of humans.

Recombinant DNA (rDNA), also called as in vitro recombination, is a procedure concerned with creating and purifying the genes of interest. Gene cloning (molecular cloning) involves generating recombinant DNA and introducing it into a host cell that is replicated. One of the fundamental strategies of molecular cloning is to move genes of interest from a complex and large genome to simple, small one. The method of in vitro recombination makes it possible to cut different strands of DNA, outside the cell (in vitro), with the aid of a restriction endonuclease and link the DNA molecules together by means of complementary base pairing.
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#3
Recombinant DNA (rDNA) technology is an area of molecular biology in which researchers edit DNA to create novel synthetic molecules, which are usually designated as chimeras. The isolation and manipulation of genes in rDNA technology facilitates more accurate genetic analysis as well as its useful applications in medicine, agriculture, industry and medicine.

The initial step in the transformation procedure of rDNA technology involves inserting a piece of DNA into a suitable vector. A molecule of DNA that carries foreign DNA into a host cell (generally yeast or bacteria), where it replicates, generating numerous copies of itself together with the foreign DNA, is known as a cloning vector. The unique features that are essential to be a cloning vector requires presence of specific sequences that allows the propagation of the vector in bacteria or yeast, presence of a cloning site to insert the foreign DNA (most of the handiest vectors contain a site that can be cleaved by many of the restriction endonucleases) and a process to select for bacteria or yeast harbouring a vector with foreign DNA (this is typically done by selectable markers for various drug resistance). The various types of cloning vectors utilized are Plasmids, Phages, Cosmids, Bacterial Artificial Chromosomes (BAC) and Yeast Artificial Chromosomes (YAC).

The genetic modifications in gene therapy can be carried out in vitro or straightforwardly in vivo by utilizing vectors that are capable of genetic transfer. Presently the most efficient procedure for gene transfer is to utilize viral vectors. They act by infecting the target cell and deliver the therapeutic gene by their usual biological phenomenon. Viral vectors that are utilized in gene therapy must be incapable to replicate and should have no lytic ability. A virus that carries an altered or foreign gene is termed as a viral vector. Some of the viruses that are utilized as vectors in rDNA technology include Retroviruses, Adenoviruses, Parvovirus, Herpes virus and Poxviruses.
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#4
The recombinant DNA technology (rDNA) is utilized mainly for two types of cloning namely reproductive and therapeutic cloning. Reproductive cloning will create a life form with the same genetic information of the one that exists already. This technique has been already carried out with some animals. A sheep known as Dolly was the first mammal to be reproduced as a precise genetic copy. Therapeutic cloning is utilized in the reproduction of certain tissues or organs and not a complete organism.

Utilization of recombinant DNA technology for the purpose of therapeutic cloning has immense deal of benefits. For instance, an organ that is cancerous could be replaced with a new one prepared from the own DNA of the patient. This would possibly help to minimize the rejection of organs that occurs occasionally when a transplant of the organ is carried out. If an organ like heart is injured, it could even be replicated with the aid of this technology. Despite the fact that these applications may be many years from practical use, they are future possibilities.

Some of the examples of the therapeutic products prepared from recombinant DNA techniques include blood proteins (Erythropoietin, Factors VII, VIII, IX, Tissue plasminogen activator and Urokinase), human hormones (Epidermal growth factor, Follicle stimulating hormone, Insulin, Nerve growth factor, Relaxin, Somatotropin), immune modulators ( Interferons, Colony stimulating factor, Lysozyme and Tumor necrosis factor) and vaccines (Cytomegalovirus, Hepatitis B, Measles and Rabies).
The rDNA techniques of gene alteration, cloning and expression were initially developed in bacteria but are now applied functionally in a diversity of model eukaryotes. Even though eukaryotic genes are cloned and sequenced with the aid of bacterial hosts, it is often advantageous to introduce such genes back into the original eukaryotic host or into another eukaryote to create a transgenic eukaryote. The likelihood of transgenic modification of eukaryotes such as plants and animals (also humans) creates many novel research opportunities.
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