Application of genetic engineering in medicine pdf

One approach to obtaining a clone of a gene is to isolate it from a genomic library through the use of a specific probe. There are three ways to produce genomic libraries: 1. Genomic DNA is completely digested by a restriction enzyme, and the resulting DNA fragments are then cloned in a cloning vector.

This technique does have a drawback. If the specific gene the researchers want to study contains restriction sites for the enzyme, the gene will be split into two or more fragments when the DNA is digested by the restriction enzyme.

In this case, the gene would then be cloned in two or more fragments. Thus, an entire library would need to contain a very large number of recombinant DNA molecules, and screening for the specific gene would be very laborious. The problems of genes split into fragments and the large number of recombinant DNA molecules can be minimized by cloning longer DNA fragments. For example, the passage of the syringe needle will produce a population of overlapping DNA fragments. However, since the ends of the resulting DNA fragments have not been generated by cutting with restriction enzymes, additional enzymatic manipulations are necessary to add appropriate ends to the molecules for insertion into vector cloning sites.

Another approach for producing DNA fragments of appropriate size for constructing a genomic library is to perform a partial digestion of the DNA with restriction enzymes that recognize frequently occurring four-base —pair recognition sequences. Partial digestion means that only a portion of the available restriction sites is actually cut with the enzyme. The ideal result of partial digestion is a population of overlapping fragments representing the entire genome.

Sucrose gradient centrifugation or agarose gel electrophoresis is then used to collect fragments of the desired size for cloning. Those fragments can be cloned directly since the ends of the fragments were produced by restriction enzyme digestion. The recombinant DNA molecules produced by this method are used to transform E.

In the case of plasmid and cosmid libraries, the transformants are plated on selective medium to clone the sequences. Each colony that is produced almost always represents a different cloned DNA sequences since each bacterium that gave rise to a colony most likely contained a different recombinant DNA molecule.

The aim of this method to produce a library of recombinant molecules that is as complete as possible. However, not all sequences of the eukaryotic genome are equally represented in such a library, for example, if the restriction sites in a particular region are very far apart or extremely close together, the chances of obtaining a fragment of clonable size are small.

In 1-f molecules , P is the probability desired, and f is the fractional proportion of the genome in a single recombinant DNA molecule. Suppose that we wish to prepare a library of human genome using a lambda phage vector. The human genome contains 3. This makes searching the library for a gene of interest very time consuming. One approach for reducing the searching time of large genomes is to make libraries of individual chromosomes in the genome.

In humans, this gives 24 different libraries, one each for 22 autosomes, the X and the Y. Then, if a gene has been localized to a chromosome by genetic means, researchers can restrict their attention to the library of that chromosome when they search for its DNA sequence. Individual chromosomes of an organism can be separated if their morphologies and size are distinct enough, as is the case for human chromosomes.

One procedure currently used to isolate large chromosomes individually is flow cytometry. The stained chromosomes flow passed a laser beam connected to light detector.

This system sorts and fractionates the chromosomes based on their differences in dye binding and resulting light scattering. Approximately chromosomes can be sorted and isolated per second. Once the chromosomes have been fractioned, a library of each chromosome type can be made by cutting the chromosomal DNA with restriction enzymes and inserting the fragments into cloning vector. As a result of the application of these procedures, libraries of DNA prepared from all human chromosomes are now available to researches.

These cDNA molecules can be cloned. More typically, the entire mRNA population of a cell is isolated and a corresponding set of cDNA molecules is made and inserted into a cloning vector to produce a cDNA library. Since a cDNA library reflects the gene activity of the cell type at the time the mRNAs are isolated, the construction and analysis of cDNA libraries is useful for comparing gene activities in different cell types of the same organism, because there would be similarities and differences in the clones represented in the cDNA libraries of each cell type.

In eukaryotes, mature mRNAs are processed molecules having no introns, so the sequences obtained are not equivalent to gene clones. So gene clones are much more informative than can cDNA clone, for example, on the presence and arrangement of introns, and on the regulatory sequences associated with gene.

However, the cloning of DNA is time consuming, involving the insertion of DNA into cloning vectors and typically the screening of libraries to detect specific DNA sequences.

In the mid- s, the polymerase chain reaction PCR was developed and this has resulted it yet a new revolution in the way genes may be analyzed. PCR is a rapid cell-free method for producing an extremely large number of copies of a specific DNA sequence from a DNA mixture without having to clone it, a process called amplification.

In brief, the PCR procedure is as follows: 1. Denaturing the DNA to single strands by incubating at 94oC. Cool to C, depending on how well the base sequences of the primers matches the base sequence of the DNA and anneal the specific pair of primers primer A and primer B that flank the target DNA sequence. Extend the primers with DNA polymerase. Repeat the heating cycle to denature the DNA to single strands and cooling to anneal new primers.

Repeat the primer extension with Taq DNA polymerase. In each of the two double stranded molecules produced, one strand of unit length; that is, it is the length of the DNA between the 5-prime end of primer A and the 5-prime end of the primer B- the length of target DNA. The other strand of both molecules is longer than unit length.

Repeat the denaturation of DNA and anneal the new primers. Repeat the primer extension with Taq polymerase. This produces unit-length, double—stranded DNA. Note that it took three cycles to produce the two molecules of target-length DNA. Repeated denaturation, annealing, extension cycles results in geometric increase of the unit- length DNA. Amplification of longer-than- unit- length DNA occurs simultaneously, but only in a linear fashion. Starting with one molecule of DNA, one cycle of PCR produces two molecules, two cycles produces four molecules, and three cycles produces eight molecules, two of which are the target DNA.

A further ten cycles produces 1, copies 2 10 of the target DNA and in 20 cycles there will be 1,, copies 2 30 of the target DNA! The procedure is rapid, each cycle taking only a few minutes using a termal cycler, a machine that automatically cycles through the temperature changes in a programmed way.

Only the disadvantage of the PCR procedure is that Taq polymerase does not have proof reading properties, so that errors are introduced into the DNA copies at low frequencies. Of course, should an error be introduced in an early cycle of PCR, then all subsequent copies made from that DNA would have that error.

PCR also susceptible to contamination. There are many applications for PCR, including amplifying DNA for cloning, amplifying DNA from genomic DNA preparations for sequencing without cloning, mapping DNA segments, disease diagnosis, sex determination of embryos, forensics, studies of molecular evolution, etc.

In each case, some DNA sequence information must be available so that appropriate pairs of primers can be synthesized. Some practical applications are as follows: Medical applications: Certainly the production of medically useful proteins such as somatostatin, insulin, human growth hormone, and interferon are of great practical importance. This is particularly true of substances that previously only could be obtained from human tissues.

Interleukin-2 a protein that helps regulate the immune response and blood clotting factor VIII have recently been cloned, and undoubtedly other important peptides and proteins will be produced in future. It also be possible to use genetically engineered plants transgenic plants to produce oral vaccines. A recombinant hepatitis B vaccine is already commercially available.

Probes are now being used in the diagnosis of infectious diseases. An individual could be screened for mutant genes with probes and hybridization techniques even before birth when used together with amniocentesis. A type of genetic surgery called somatic cell gene therapy may be possible for artificial individuals.

For example, cells of individual with a genetic disease could be removed, cultured and transformed with cloned DNA containing a normal copy of yhe defective gene s. These could then be reintroduced into the individual; if they become established, the expression of normal genes might cure patients. Recently so many techniques have been developed by different researchers including the virosome model, the most efficient technique of gene delivery, developed by Dr. Sarkar of Delhi University to transfer correct genes to the patient.

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Related titles. Carousel Previous Carousel Next. Production of entomopathogenic nematodes in submerged monoxenic culture: A review. These products are so-called GMOs genetically modified organisms or Transgenic. Genetic engineering has gained significant importance in the production of medicines. At present, plants and microorganisms that form the basis of certain drugs are being genetically modified to create better vaccines, more effective treatments, enzymes or hormones at low cost.

Medical research has received from the genetic engineering the knowledge necessary to identify genes that produce catastrophic or incurable diseases.

These genes can be diagnosed early and cured or avoided, depending on the case. Gene therapy is a technique that allows the isolation of healthy genes to insert directly into people who have diseases caused by genetic malformations, thus achieving effective treatments. This therapy is, perhaps, the most promising and revolutionary contribution of genetic engineering today.

Cystic fibrosis, muscular dystrophy, hemophilia, cancer or Alzheimer's , Are some of the human ailments that are being effectively combated from their microcellular origin. Genetic recombination technology is having a high impact on energy production. Every year, huge amounts of biofuels are produced from oilseed rape Every day in the world's supermarkets, hangers are filled with products developed from genetically altered organisms.

The food industry has found in genetic engineering a way to lower costs, increase production and find new products made through genetic research. Scientists hope to be able to genetically engineer bananas to have vaccines in them.

The bananas would then be grown in developing countries, where disease such as cholera and diarrhea are very prevalent. This would be a much cheaper alternative to the wasteful process of a series of shots, throwing away costly syringes after every injection. About million people are carriers of hepatitis B, which can cause liver failure and liver cancer.

Diarrhea is a common cause of death in young children. Stem cells are cells that are basically a blank slate and can become any other type of cell. They can be found in both embryos and adults. Scientists take the stem cells, put in healthy, normal DNA, and then put them into patients to replace their cells that have defective DNA.

Manipulating stem cells is probably one of the most recognizable form of genetic engineering in medicines. Bone marrow cells of the child after removal from the body were invaded by a harmless virus into which ADA has been inserted. Genetically engineered tissue plasminogen activator tPA enzyme dissolves blood clots in people who have suffered heart attacks. The plasminogen activator protein is produced by genetech company which is so potent and specific that it may even arrest a heart attack underway.

Antibodies cloned from a single source and targetted for a specific antigen monoclonal antibodies have proved very useful in cancer treatment.

Monoclonal antibodies have been target with radioactive elements or cytotoxins like Ricin Ricin is a highly toxic, naturally occurring lectin produced in the seeds of the castor oil plant, Ricinus communis. A dose of purified ricin powder the size of a few grains of table salt can kill an adult human.

Such antibodies seek cancer cells and specifically kill them with their radioactivity or toxin. Anukriti Xalxo Dec. Kelly Jones Nov. Kondashalinisubraman Nov.

Rashmi Barethiya Sep. Md Shamshad Aug. Show More.