Genetics is a very fast growing field in the 21^st century.Genetic engineering and biotechnology has revolutionised medical science.Now every disease has got a genetic base, and the scope and research in this field are getting more and more wide.Days are not far when genetically modified food will be used in our homes.There are many application of this technology, but the base of everything related to genetics lie on DNA, genes and it manipulation.Gene therapy which was once thought to be a fantasy has become a reality today.Much more advancement has taken place in many fields of medical science by the introduction of biotechnology and genetic engineering.In this article I am considering the most basic concept of biotechnology which is called recombinant DNA technology and its greatest application called Gene therapy.

What is Recombinant DNA Technology?

Before we understand recombinant DNA technology, we have to know about biotechnology.Biotechnology may be defined as “The method by which a living organism or its parts are used to change or to incorporate a particular character to another living organism”.When DNA is used for incorporating a character it is called Genetic recombination.In other words Genetic recombination is the exchange of information between two DNA segments.This occur commonly with the same species.But when artificial means are used, in which a gene of one species is transferred to another living organism, it is called recombinant DNA technology.In common parlance, this is known as genetic engineering.In a common man's words, it can be said as cutting a gene or a part of DNA from one organism and pasting it to the DNA of another so that the character given by the gene is expressed in the organism.An example of this process is the cutting of gene coding for insulin from human beings and pasting it into an E-coli bacteria, so that E-coli start to produce insulin.Thus whichever character or substance we need, the corresponding gene can be isolated and pasted in a new organism in order to get that product in large scale.

Application of Recombinant DNA Technology

Gene therapy is considered to be one of the most important application of recombinant DNA technology.Many researches are going on and presently gene therapy is in its developing stage.In gene therapy, a normal gene is used to substitute the work done by the abnormal gene responsible for the disease.The other most important application is quantitative preparation of Bio molecules.For example if we want to get one unit of growth hormone, we have to take more than 1000 pituitary glands from the dead body, which is not at all practical.But using recombinant DNA technology we can get large amount of such hormones or any other biological products.The genes responsible for the production of these substance can be isolated and incorporated to other organisms like bacteria and thus the product can be obtained in large scale.In the same way, it is also possible to produce a biologically important substance without any contamination.For example lets take the case of Hepatitis B vaccine.The hepatitis B virus vaccine is prepared by using hepatitis B virus surface protein, which will give protection from infection.In early times, to obtain this surface proteins, the blood of the patients was used and the proteins was isolated.But the blood of hepatitis B person is highly infective.So when we make the vaccine we can’t rule out the possibility that the sample is contaminated with hepatitis B virus particle.But when Recombinant DNA technology is used, we can produce that virus surface protein by isolating the gene coding for the protein and thus can be prepared in large scale.This method ensures that no virus particle is involved and the product is 100% safe.

In the above step we have isolated genes under recombinant DNA technology and such isolated gene can also be used as specific probes for diagnosis of diseases.Probes means a known part of DNA labelled with radioactive substance which can be used in various studies.There are many uses of this probe which include, diagnosis of a genetic disease, to identify viral particles or bacterial DNA in suspected blood and other tissue samples, to demonstrate virus integration in transformed cells, to detect activation of oncogenes in cancer (proto oncogenes transform into oncogene and cause cancer), to pinpoint the location of gene in a chromosome and also to identify mutation in genes.Some other products made by using this technology are human proteins like interleukins, interferons, anti-hemophilic globulin, hormones etc..These are only some of the examples and day to day we are getting more and more application of this technology.

Tools of Recombinant DNA Technology

The most important and basic tool of recombinant DNA technology is called Restriction Endonuclease (RE).We know that in this technology, in order to transfer a gene first the gene has to be cut from the parent DNA.This is possible using Restriction endonucleases.They will identify specific sequence or part in DNA and will excise that part.Thus restriction endonuclease is referred to as molecular scissors.It was a scientist named Werner Arber who showed that certain enzyme in bacteria restrict the entry of the phage into host bacteria.Phage is a DNA like substance or organism.It was a defence mechanism of bacteria where it produce RE to cut the incoming phage and destroy it.Thus because of its mode of action, it is named as Restriction endonucleases.Hamilton Smith isolated the first restriction enzyme called beta hind I and it was Paul Berg who developed the cutting technique for recombinant DNA technology.In this technology what restriction endonucleases does is that, it identify specific region in DNA and they cut the DNA in that specific region.Now there are more than 800 such enzymes available commercially.These enzymes recognize specific sequence with palindromic arrangement.Palindrome in Greek means ‘to turn backwards’.It is similar to a word that can be read same in both forward and backward direction.Eg-madam.But here the words are in form of genetic letters of life represented by four letters A, T, C, G which stands for Adenine, Thymine, Cytosine, Guanine respectively.Thus by identifying a gene and locating a palindromic sequence on both ends of the gene, we can apply the correct restriction endonuclease for the sequence and cut that part from DNA.Here we must understand that though we use the terms cut and paste the actual process is very complicated.So now we have got the gene.This gene has to be incorporated into the recipient organism.To insert or paste the gene into the recipient organism, we need another tool which is called a Vector.A vector acts as a carrier of the gene and help in the introduction of the gene.The most commonly used vector are called Plasmids.Plasmids are circular double stranded DNA molecules seen inside bacteria.They can replicated independent of bacterial DNA.Here in order to insert the foreign DNA into the vector, we have to use restriction endonucleases.Other examples of vectors are Bacteriophage and Cosmids.

Procedure of DNA Recombination.

The first step of DNA recombination is the preparation of specific human gene.But to isolate a specific gene from human DNA is a laborious process.So to get the gene, we have to find the m-RNA for that gene, which can be easily found in the cell which express that gene.m-RNA is a complementary copy of the gene.For example, to get the gene coding for the insulin, we just have to get the m-RNA from the islets of Langerhans cells from the pancreas.Once m-RNA is isolated, we can make a complementary copy of DNA or c-DNA using the enzyme reverse transcriptase.Thus the resultant DNA formed will be an exact copy of the actual gene.Now the gene is isolated and we have incorporate it into a vector.Such a vector carrying a foreign DNA is called Chimeric DNA.To make the chimeric DNA we use all the enzymes like restriction endonucleases and DNA ligase etc..We have already discussed the working of RE.DNA ligase enzyme help in the formation of phosphodiester linkage between vector and the insert DNA part.Thus a chimeric DNA is finally produced.The next step is the cloning of chimeric DNA.A clone is a large population of identical bacteria or cell that arise from a common ancestor.By cloning we get a large number of identical DNA molecules.Thus the hybrid chimeric DNA are amplified by cloning technique.

Now we have to use the large amount of plasmids to change the normal bacteria.The process by which the plasmid is introduced into the host is called Transfection.The host bacteria eg-E.coli and plasmid vector are incubated in hyper tonic medium containing calcium for few minutes.Here what happens is that ion channels in host cell or E.coli is opened and plasmid will enter the host through such channels.After tranfection, the host cells are allowed to grow in agar plates containing growth medium.But here we can't expect that every E.coli is transformed by the entry of vector.Only about 5% of bacterial colonies may contain the desired vector.So next step is to select the desired colony.To differentiate the hybrid DNA and other normal DNA, we use the property of antibiotic resistance in the bacteria caused by the plasmid.For this we use plasmid which has got genes resistant to antibiotics.The foreign DNA will be inserted into the plasmid in such a way that, one of the antibiotic resistance in plasmid is lost due to presence of this foreign DNA in the middle.Once this plasmid is inside the bacteria, that group of bacteria will have resistance to antibiotics except for the antibiotic resistance which was lost due to the insertion of foreign DNA.This insertional inactivation of antibiotic resistance in plasmid via bacteria can be used as marker in identifying the transformed or hybrid bacteria.

To make it more clear lets take an example of a plasmid called pBR-325 which has got antibiotic resistance to three antibiotic namely, ampicillin, tetracycline, chloramphenicol.The restriction endonuclease will cleave the plasmid in the middle of chloramphenicol gene and the foreign DNA is inserted there.After tranfection the bacteria are cultured in a medium containing ampicillin and tetracycline.The antibiotic will kill the wild type or normal bacteria and only the bacteria with plasmid will survive.So we get a group of bacteria containing the plasmid.But still its not necessary that this plasmid has got the foreign DNA fixed in it.There can be some bacteria which have not received the foreign DNA and thus not hybrid.In that case, we have to select some bacteria from the colony and put it in chloramphenicol.Since the hybrid bacteria don’t have resistance to choramphenicol due to insertional inactivation, they will die.So we have to watch which bacteria died and isolate the corresponding colony from which it was taken.So that colony of bacteria is our hybrid bacteria.They are transferred to other dish and cultured to produce clones.Thus a large number of bacteria which express the gene we required is formed and can be used for mass production and commercial production.These bacteria will produce the substance coded by the gene, mainly proteins which can be further processed to get the products we want.To produce human proteins such plasmid inside E.coli is used without any protein inhibitor and thus proteins are obtained, and such vectors are called Expression Vector.

GENE THERAPY

As I said in the introduction, gene therapy was once considered as a fantasy.It is the most wonderful application of recombinant DNA technology.The first breakthrough in gene therapy came when a girl who was suffering from Severe Combined Immunodeficiency was treated with gene therapy.Here the patient had deficiency of Adenosine Deaminase enzyme and it was treated by transferring the normal gene for adenosine deaminase.In gene therapy the genes are delivered into a cell to generate a therapeutic effect by correcting the existing abnormality.Here the abnormal gene may or may not  be replaced, but the healthy gene is introduced.Currently only somatic gene therapy is done, and germ cell gene therapy is considered as unethical.

Procedure of Gene Therapy

Just as we discussed in recombinant DNA technology, its all about isolating a healthy gene with all the parts needed for expression, then incorporation the gene into a carrier or vector and finally deliver the vector to the target cell.There are three ways by which gene can be introduced.First method is called Ex Vivo strategy where the cells of patients are cultured in laboratory, new genes are introduced into that cells and these modified cells are introduced back into the patient.The second method is just opposite to this where the vector is administered directly to the cell.It is called In Vivo strategy.One example where this method is used is in cystic fibrosis where the vector is introduced into respiratory tract cells.The third method is called In situ strategy where expression vector is injected to the patients either intravenously or directly to the tissue.Once the normal gene reaches the cell, it get incorporated in DNA or work along with the DNA producing the deficient substance in body.Thus normal condition can be attained and disease can be cured.

The vectors mainly used for this purpose are Retroviruses, Adenoviruses, adeno associated virus particles, and herpex simplex viruses.Non-virus system include liposomes plasmids and physical methods.Retrovirus are actually RNA virus that replicate through DNA intermediate and they will have enzyme called reverse transcriptase.Moloney Murine Leukemia Virus (MMLV) is commonly used.What is done with retro virus is that, the disease causing and replicating part of the RNA will be replaced by the normal gene we want to get introduced.The virus protein covering also will be fixed to the virus in other means.Its a total genetic engineered product.This replication deficient ineffective virus is then introduced into the cells of patient.Inside the cell, this virus will produce the DNA copy of the gene and just like it infect the host DNA it will introduce the gene into the host DNA.Here in actually case the virus DNA was to be introduced, but since we have replaced it by genetic engineering, now the desired DNA is getting introduced.Thus the gene start working in the host DNA and the disease is cured.The main advantage of this method is that, since the virus is engineered, its very safe and also this virus can infect many types of cell.This method is best when we consider disease produced by single gene mutations.But the main disadvantage is that only dividing cells can be the targets of retrovirus and also less amount of virus can be produced in this way.There is also a theoretical chance that the virus again be infectious by mutating back to the original form.

One of the other system used for the same purpose is Adeno virus.The main difference is that adeno virus are DNA virus.When the virus with the human gene reach the nucleus of the cell, it wont integrate the gene into the host DNA but will remain as epichromosomal particle.It will be separate from the host DNA and it will express the gene.The main advantage is that it can be made in large numbers and it will easily infect a host cell.But the expression will be for a very little time, maybe weeks or months.There is still another system for carrying gene which is called Plasmid Liposome complex.It is a non-viral vector system.Liposomes are artificial lipid bi layer and form  complex with plasmid carrying normal human DNA.Because the complex is lipid, it can easily pass the plasma membrane and positive charge in the complex will spontaneously attach to negative charge DNA.The main advantage is that large genes can be transferred and it wont replicate or evoke any immune response.But there is a disadvantage that most of these complex can get destroyed in host cell and thus majority of cases can be unsuccessful.So if this is not the method preferred ,then a different form of non-viral gene transfer can be considered which is called as Gene gun.As the name implies, it  is a particle gun and plasmid DNA coated with tungsten particles are literally shot into the cell using helium pressure discharge.This help the particles to reach the target tissue and its very fast and can be done in most cells.But cellular damage and transient gene expression are the drawbacks.In general, though gene therapy can be used to treat many disease,at the same time it has got many limitations like inconsistent result, lack of ideal vector, sometimes death during the course of gene therapy due to some enzyme deficiency, reactivation of retro virus leading to leukemia etc.It will take many more years before gene therapy is fully successful

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So with recombinant DNA technology, a new revolution has come in the medical field.With gene therapy the days are not far when there is cure to every genetic disease.Presently gene therapy is most effective in treating inherited disorders caused by single gene.It will take decades before gene therapy can be used as a common clinical procedure.But day by day the application of recombinant DNA technology are increasing and we already enjoy many of its fruit.