DNA-deoxyribonucleic acid is present in nuclei of cells which makeup the body of the organism. It is considered as one of the building block of the body. It is a molecule encoding genetic instructions used in functioning and development of all living organism and viruses. DNA is the one of three major macromolecules along with RNA and proteins. Genetic information is encoded as a sequence of nucleotides that are added in guanine, adenine, thymine and cytosine represented as G,A,T and C. DNA are molecules with double stranded helices consisting of two long polymers of units called nucleotides. Molecules with backbones made of alternating sugar and phosphate group with the nucleobases, attached to sugar. Deoxyribonucleic is well suited for biological information storage. This double stranded provides the molecule with built-in duplicate of encoded information and resistance cleavage.
The two strands run in opposite direction to each other, one from three prime to five prime ends and other from five to three prime ends. They actually represent the fifth and third carbon on sugar molecule. Within a cell, DNA is organized in a long structure called chromosome.
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WHAT IS GENE
It is a molecular unit of heredity of living organism. Actually it is a name given to some stretches of DNA and RNA that codes for RNA chain or code for polypeptide that has function in organism, living being depends on gene as they specify all proteins and functional RNA chain,
Gene held information pass genetic traits in offspring and to maintain an organism's cell. All organisms have many genes, some are visible like eye color or limbs number and some are not visible such as blood type, increase risk for disease.
Gene work as a subunit of DNA and each gene contain a particular set of instructions, usually coding for a particular protein are for a particular function
Recombination is the exchange of corresponding DNA segments between adjacent chromosomes during special type of cell division that result in production of new genetic material.
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA molecules that result from bring together genetic material from multiple sources.
To join together DNA molecule from two different species that are inserted in host organism, to produce new genetic combination that have value to science. This is also known as genetic engineering.
Two scientists named Boyer and Cohen in 1973 developed this recombinant DNA technology.
Firstly prepare the chromosome DNA and then desired gene fragment is cut by restrictive endonuclease and then added to vector (host plasmid) by creating sticky ends. This is recombinant DNA.
COMONENTS INVOLVED IN DNA TECHNOLOGY:
â€¢ Gene of interest
â€¢ Antibiotic resistance gene
â€¢ Antibiotic resistance marker
â€¢ Endonuclease enzymes
Restriction endonuclease is an enzyme that recognizes the DNA at special nucleotide sequences called as restriction sites.
Such enzyme is found in archaea and in bacteria and their function is to defend against viruses.
More than 3000 restriction enzymes are studied in detail. These enzymes are used in DNA modification and are vital biological tool in molecular cloning.
Restriction enzyme, term first used by the studying phage lambda.
Another definition of restriction endonuclease is given below:
"These are the enzymes that cut the DNA at its sugar-phosphate position of backbone."
Restriction enzymes get their name after the origin of their host. For example from Escherichia Coli, Eco R1was isolated and from Haemophilus Hind II and Hind III were isolated.
It recognizes the specific sequences of nucleotides and produced a cut in the double stranded DNA. These sequences may vary from 4 to 8 nucleotides but many of these are palindromic sequences, it means that whether we read the base sequence from 5` end to 3` end or either from 3` end to 5` end, the sequences were same. For example GATATC sequence is complementary to CTATAG.
When different restriction enzymes read the same nucleotide sequences are called as NEOSCHIZOMERS.
USES OF RESTRICTION ENZYMES
They are used in manipulation of DNA. They are also used to insert genes in the vectors (i.e plasmid) while doing gene cloning.
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Restriction enzymes are used in digestion of genomic DNA in southern blotting technique. These are also used in RFLP (restriction fragment length polymorphism)
In molecular genetics, the vector is a vehicle that is used to carry genetic information and inserted into the target cell, where it is replicated and expressed. There are four major types of vectors: plasmids, cosmids, viral vectors and artificial chromosome.
Plasmids should be self replicating in a host cell. Plasmids have many features, especially including multiple cloning sites, which contains overhangs for the foreign gene to insert.
CLONING OF DNA:
Cloning of DNA involves the taking of chromosomes and then obtaining gene or DNA which contain that gene and attaching that segment into the carrier DNA and then replication of this DNA to many times such as millions of time.
ANTIBIOTIC RESISTANT GENES:
There are many genes that are used in the DNA recombinant technique for antibiotic resistance such as
1: Amphisaline resistance genes these are:
A: PVU 1
B: PST 1
2: Tetracycline genes:
BASIC PRINCIPLES INVOLVED IN DNA RECOMBINANT TECHNOLOGY:
First step in the principles of recombinant technology is that taking gene of interest and gene amplification.
Second step is the vector construction in which different vectors are used like virus, bacteriophages, bacterias etc are used. They are called vectors because vector is a vehicle that transports things from one place to another place and these vectors also do that. Plasmids are used because they are responsible of self replicating. Recombinant vector is produced in which foreign gene is introduced in the plasmid in which gene is amplified restriction enzymes are used they create sticky ends in the gene and plasmid. Same restriction enzyme is used otherwise gene and plasmid will not liquate after that legate enzyme is used which legate gene of interest and plasmid now our recombinant plasmid has been used.
APPLICATIONS OF DNA RECOMBINANT TECHNOLOGY:
There are many applications of DNA recombinant technology or genetic engineering. Applications of genetic engineering involves modification of individuals, preparation of different products of industries and production of crops that are resistant to many diseases and insect pests like B-T cotton etc. Genetically modified plants are being produced by this method like GMO'S. Vaccines are being produced by genetic engineering.
1: Control of feral pests: In this technique resistance against a virus known as myxomatosis in the rabbits has been produced mostly in the Europe this technique is applied.
2: PRODUCTION OF HUMAN INSULIN:
1: Taking of human insulin gene: In this technique gene is obtained. First of all take DNA and form RNA and then by reverse transcriptase enzyme make COMPLEMENTRY DNA (cDNA).
2:PREPARATION OF PLASMID: Plasmid is prepared by taking it from any source then is amplified
3: JOINING OF THE GENE INTO THE VECTER PLASMID:
Sticky ends are created on the gene of interest and the plasmid by the restriction enzyme. Same restriction enzyme is used for this cutting and creating sticky ends. Next step is the ligation of gene into the plasmid on the sticky ends and at the last ligase enzyme is used for ligation of ends.
4:INTRODUCTION OF THE RECOMBINANT PLASMID INTO THE BACTERIA:
That recombinant plasmid is used to introduced into the host bacteria E coli is used as a host because it will replicate rapidly. After introduction of plasmid into the host cell as we have introduced into the E.coli. For this porpose test tube is taken place and temperature of 42c is given in which there is recombinant pasmid and the host organism that is the E coli at this temperature the recombinant plasmid will enter into the E coli.
5:SELECTION OF THAT BACTERIA THAT HAS TAKEN THE CORRECT PART OF DNA:
For this purpose agar medium is used for the spreading of bacteria. This agar contain antibiotics in which only those bacteria will grow that will transformed and other bacteria that are transformed will die.
VACCINE DEVELOPMENT :
There are four species of malaria has been transferred to E-COLI , one of them is surface antigen of PLASMODIUM FALUCIPARUM to produce a vaccine against this form of those who visit malaria for short period of time ..
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GENE THERAPY FOR GENETIC DISEASES :
In gene therapy it may be possible to transfer normal gene with sickle _cell anemia
Through marrow stem cells . the goal is to produce enough normal hemoglobin ..
SAFETY ISSUES IN RELATION TO RECOMBININANT DNA TECHNOLOGY :
ïƒ˜ In recombinant DNA work , bacteria is used . there is always a possibility may a pathogen
Attach with recombinant bacteria by accident and escaped from the laboratory and May cause epidemic for which no drugs were available ..
ïƒ˜ Recombinent DNA Advisory Committee was established in 1974 in the United States , which work on the safety of manipulation of genetic material â€¦
DANGEROUS OF DNA RECOMBINENT TECHNOLOGY :
It is always possible that may antibiotic _resistance plasmid incorporatedinto a dangerous pathogen with medical consequence ..
Genetically modified crops
Genetically modified crops (GM crops, or biotech crops) are plants, the DNA of which has been modified using recombinant DNA technology, to resist pests and agents causing harm to plants and to improve the growth of these plants to motivate farmers efficiency.
Genetic engineering techniques are much more accurate than mutagenesis (mutation breeding) where an organism is exposed to radiation or chemicals to create a non-specific but stable change. Other techniques by which humans modify food organisms include selective breeding; plant breeding, and animal breeding.
In most cases the aim is to introduce a new desirable traits to the plant which does not occur naturally in this species. Examples include resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent.
Gene flow in plants
Scientists first discovered that DNA naturally transfers between organisms in 1946. It is now known that there are many natural mechanisms for gene flow and that these occur in nature on a large scale - for example, it is a major mechanism for in pathogenic bacteria, and it occurs between plant species. This is facilitated by transposons, rrtrotrasposons, proviruses that naturally translocate to new sites in chromosomes. They often move to new species over an evolutionary time scale and play a major role in dynamic changes to chromosomes during evolution.
The introduction of foreign gene into crops has been achieved by traditional crop breeders by artificially overcoming fertility barriers. A hybrid cereal was created in 1875, by crossing wheat and rye. Since then important traits have been introduced into wheat, including dawarfting gene and rust resistance.
Improved shelf life
Production of biofuels
Some important GM crops
Genetically modified (GM) cotton was developed to reduce the effect of pesticides. The bacterium Bacillus thuringiensis (Bt) naturally produces a chemical harmful only to a small fraction of insects, most notably the larvae of moth, butterflies, beetles, and flies, and harmless to other forms of life. The gene coding for Bt toxin has been inserted into cotton, causing cotton to produce this natural insecticide in its tissues. In many regions, the main pests in commercial cotton are lepidopteron larvae, which are killed by the Bt protein in the transgenic cotton they eat. This eliminates the need to use large amounts of broad-spectrum insecticides to kill lepidopteron pests.
Rice produces Î²-carotene in the leaves but not in grains, where the biosynthetic pathway is turned off during plant development. In Golden Rice two genes have been inserted into the rice genome by genetic engineering, to restart the carotenoid biosynthetic pathway leading to the production and accumulation of Î²-carotene in the grains. The intensity of the golden colour is an indicator of the concentration of Î²-carotene in the endosperm.
Bt-corn is a type of genetically modified organism, termed GMO. A GMO is a plant or animal that has been genetically modified through the addition of a small amount of genetic material from other organisms through molecular techniques. Currently, the GMOs on the market today have been given genetic traits to provide protection from pests, tolerance to pesticides, or improve its quality. Examples of GMO field crops include Bt-potatoes, Bt-corn, Bt-sweet corn, Roundup Ready soybeans, Roundup Ready Corn, and Liberty Link corn
A donor organism may be a bacterium, fungus or even another plant. In the case of Bt corn, the donor organism is a naturally occurring soil bacterium, Bacillus thuringiensis, and the gene of interest produces a protein that kills Lepidoptera larvae, in particular, European corn borer. This protein is called the Bt delta endotoxin. Growers use Bt corn as an alternative to spraying insecticides for control of European and southwestern countries.
Soybeans have been developed that express a crystalline insecticidal protein from Bacillus thurigienesis
Bt Soybeans are genetically modified that produce an insecticidal protein like the one naturally produced by the bacteria species Bacillus thurigienesis.
The Bt brinjal is a type of genetically modified brinjal (also known as an eggplant) created by inserting a crystal protein gene(Cry1Ac) from the soil bacterium Bacillus thuringiensis into the genome of different brinjal cultivers.. The Bt brinjal has been developed to give resistance against lepidoptaron insects, in particular the Brinjal Fruit and Shoot Borer.
A genetically modified tomato, or transgenic tomato is a tomato that has had its genes modified, using recombinant DNA technology. The first commercially available genetically modified food was a tomato engineered to have a longer shelf life (the Flavr savr). Currently there are no genetically modified tomatoes available commercially, but scientists are developing tomatoes with new desirable traits like increased resistance to pests or environmental stresses.