Genetic Engineering

Genetic engineering refers to the use of in vitro techniques to alter genetic material in the laboratory. Such altered genetic material may be reinserted into the original source organism or into some other host organism. Genetic engineering depends upon our ability to cut DNA into specific fragments and to purify these for further manipulation. In this chapter, we will see some of the basic techniques involved in the genetic engineering.

1. Polymerase Chain reaction

The polymerase chain reaction (PCR) is essentially DNA replication in vitro. The PCR can copy segments of DNA by up to a billion fold in the test tube, a process called amplification. This yields large amounts of specific genes or other DNA segments that may be used for a host of applications in molecular biology. PCR uses the enzyme DNA polymerase, which naturally copies DNA molecules. Artificially synthesized primers are used to initiate DNA synthesis, but are made of DNA (rather than RNA like the primers used by cells). PCR does not actually copy whole DNA molecules but amplifies stretches of up to a few thousand base pairs (the target) from within a larger DNA molecule (the template). PCR was conceived by Kary Mullis, who received a Nobel Prize for this achievement.

The steps in PCR amplification of DNA can be summarized as follows:

1. The template DNA is denatured by heating.

2. Two artificial DNA oligonucleotide primers flanking the target

3. DNA are present in excess. This ensures that most template strands anneal to a primer, and not to each other, as the mixture cools.

4. DNA polymerase then extends the primers using the original DNA as the template.

5. After an appropriate incubation period, the mixture is heated again to separate the strands. The mixture is then cooled to allow the primers to hybridize with complementary regions of newly synthesized DNA, and the whole process is repeated.

The power of PCR is that the products of one primer extension are templates for the next cycle. Consequently, each cycle doubles the amount of the original target DNA. In practice, 20–30 cycles are usually run, yielding a 106-fold to 109-fold increase in the target sequence (Figure 6.24d). Because the technique consists of several highly repetitive steps, PCR machines, called thermocyclers, are available that run through the heating and cooling cycles automatically. Because each cycle requires only about 5 min, the automated procedure gives large amplifications in only a few hours.

Taq Polymerase

The original PCR technique employed the DNA polymerase Escherichia coli Pol III, but because of the high temperatures needed to denature the double-stranded copies of DNA, the enzyme was also denatured and had to be replenished every cycle. This problem was solved by employing a thermostable DNA polymerase isolated from the thermophilic hot spring bacterium Thermus aquaticus. DNA polymerase from T. aquaticus, called Taq polymerase, is stable to 95°C and thus is unaffected by the denaturation step employed in the PCR. The use of Taq DNA polymerase also increased the specificity of the PCR because the DNA is copied at 72°C rather than 37°C.

2. Restriction digestion of DNA

The second basic technique involved in genetic engineering the restriction digestion of DNA by the enzymes called “restriction endonucleases”. Restriction enzymes recognize specific base sequences (recognition sequences) within DNA and cut the DNA. Although they are widespread among prokaryotes (both Bacteria and Archaea), they are very rare in eukaryotes. In vivo restriction enzymes protect prokaryotes from hostile foreign DNA such as virus genomes. However, restriction enzymes are also essential for in vitro DNA manipulation, and their discovery gave birth to the field of genetic engineering. Restriction endonuclease (Type III) bind to the DNA at their recognition sequences and cleave the DNA within their recognition sequences, making this class of enzymes much more useful for the specific manipulation of DNA.

The restriction enzymes provide options to cut the DNA at specific site and allow to join with another DNA with the same ends. Hence, by use of these enzymes recombinant DNA formation is rather become very easy. Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome. This would be possible by the restriction digestion of the DNA.

3. Gel electrophoresis

Electrophoresis is a procedure that separates charged molecules by migration in an electrical field. The rate of migration is determined by the charge on the molecule and by its size and shape. In gel electrophoresis the molecules are separated in a porous gel. Gels made of agarose, a polysaccharide, are used for separating DNA fragments. When an electrical current is applied, nucleic acids move through the gel toward the positive electrode due to their negatively charged phosphate groups. The presence of the gel meshwork hinders the progress of the DNA, and small or compact molecules migrate more rapidly than large molecules. The higher the concentration of the gel, the more large molecules are hindered. Consequently, gels of different concentrations are used to separate molecules of different size ranges. After the gel has been run for sufficient time to separate the DNA molecules, it can be stained with a compound that binds to DNA, such as ethidium bromide, and the DNA will then fluoresce orange under ultraviolet light. DNA fragments can be purified from gels and used for a variety of purposes.

4. Molecular cloning

In molecular cloning a fragment of DNA is isolated and replicated. The basic strategy of molecular cloning is to isolate the desired gene (or other segment of DNA) from its original location and move it to a small, simple genetic element, such as a plasmid or virus, which is called a vector. When the vector replicates, the cloned DNA that it carries is also replicated. Molecular cloning thus includes locating the gene of interest, obtaining and purifying a copy of the gene, and inserting it into a convenient vector. Once cloned, the gene of interest can be manipulated in various ways and may eventually be put back into a living cell. This approach provides the foundation for much of genetic engineering and has greatly helped the detailed analysis of genomes.

Steps in Gene Cloning

1. Isolation and fragmentation of the source DNA. The source DNA can be total genomic DNA from an organism of interest, DNA synthesized from an RNA template by reverse transcriptase, a gene or genes amplified by the polymerase chain reaction, or even wholly synthetic DNA made in vitro. If genomic DNA is the source, it is first cut with restriction enzymes to give a mixture of fragments of manageable size.

2. Inserting the DNA fragment into a cloning vector. Cloning vectors are small, independently replicating genetic elements used to carry and replicate cloned DNA segments. Most vectors are plasmids or viruses. Cloning vectors are typically designed to allow insertion of foreign DNA at a restriction site that cuts the vector without affecting its replication. If the source DNA and the vector are both cut with the same restriction enzyme that yields sticky ends, joining the two molecules is greatly assisted by annealing of the sticky ends.

3. Introduction of the cloned DNA into a host organism. Recombinant DNA molecules made in the test tube are introduced into suitable host organisms where they can replicate. Transformation is often used to get recombinant DNA into cells.

Plasmids as cloning vectors

The replication of plasmids in their host cell proceeds independently of direct chromosomal control. In addition to carrying genes required for their own replication, most plasmids are natural vectors because they often carry other genes that confer important properties on their hosts. In addition to independent replication, certain plasmids have other very useful properties as cloning vectors. These include (1) small size, which makes the DNA easy to isolate and manipulate; (2) multiple copy number, so many copies are present in each cell, thus giving both high yields of DNA and high-level expression of cloned genes; and (3) presence of selectable markers such as antibiotic resistance genes, which makes detection and selection of plasmid-borne clones easier.

Example plasmid vector is pUC19, which has several features to be used as vector are as follows:

It is relatively small, only 2686 base pairs.
It is stably maintained in its host (E. coli) in relatively high copy number, about 50 copies per cell.
It can be amplified to a very high number (1000–3000 copies per cell, about 40% of the cellular DNA) by inhibiting protein synthesis with the antibiotic chloramphenicol.

Lambda Phage as cloning vector

Lambda is a useful cloning vector because its biology is well understood, it can hold larger amounts of DNA than most plasmids, and DNA can be efficiently packaged into phage particles in vitro. These can be used to infect suitable host cells, and infection is much more efficient than transformation (transfection). Phage lambda has a large number of genes; however, the central third of the lambda genome, between genes J and N, is not essential for infectivity and can be replaced with foreign DNA. This allows relatively large DNA fragments, up to about 20 kilobase pairs (kbp), to be cloned into lambda. This is twice the cloning capacity of typical small plasmid vectors.

Uses of Gene cloning

Recombinant DNA is widely used in biotechnology, medicine and research. Today, recombinant proteins and other products that result from the use of DNA technology are found in essentially everywhere. In addition, organisms that have been manipulated using recombinant DNA technology, as well as products derived from those organisms, have found their way into many farms, supermarkets, home medicine cabinets, and even pet shops. The most common application of recombinant DNA is in basic research, in which the technology is important to most current work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms. Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. Some specific examples are identified below.

Recombinant chymosin: Found in rennet, chymosin is an enzyme required to manufacture cheese. It was the first genetically engineered food additive used commercially.
Recombinant human insulin: Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, or yeast (Saccharomyces cerevisiae) which then produces insulin for human use.
Recombinant hepatitis B vaccine: Hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells.
Golden rice: A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population.
Herbicide-resistant crops: Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide glyphosate (trade name Roundup), and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries.
Insect-resistant crops: Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved.

Applications of Genetic Engineering

Genetic engineering has many commercial and practical applications. Several products have been developed through genetic engineering, which are supporting the mankind. The wide application of genetic engineering in various fields are listed here:

Microbial fermentations: The microorganisms are nowadays manipulated to produce and release the metabolites of our interest. Example: antibiotics, amino acids, hormones, insulin like medicine.

Vaccines: The genetic engineering allows to produce the antigens by introducing the viral specific gene to the yeast or E. coli like host, which can safely be used for vaccination.

Mammalian proteins: Many mammalian proteins are nowadays used as therapeutic purposes for the diseases. These are earlier being extracted from human tissues and the genetic engineering now can allow the bacteria/yeast to produce the protein in it.

Transgenic crops: By gene of interest being introduced directly to the crop; the protein produced by the gene will help the plant to resist against pest (Bt crops) is classical genetic engineering’s one of the success stories.

Environmental biotechnology: Several bacteria can detoxify the pollutants present in the environment. By identifying and cloning these genes, it is possible to develop a bioremediation system to remove the pollutants from the environment.

Gene therapy: Controlling the diseases or disorders by introducing the genes instead of drugs is known as gene therapy. Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery.

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