2° alcohols: 100°– 140 °C. B) Plasmid is an extra-chromosomal DNA molecule in bacteria that is capable of replicating, independent of chromosomal DNA. Medical ailments such as leukaemia and sickle cell anaemia can be treated with this principle. Listed below are the applications of gene cloning: - Gene Cloning plays an important role in the medicinal field. Let's understand each step more in detail. The desired genes and the vectors are cut by the same restriction enzymes to obtain the complementary sticky notes, thus making the work of the ligases easy to bind the desired gene to the vector. The deprotonated acid (the base) then reacts with the hydrogen adjacent to the carbocation and form a double bond. Clones are genetically identical as the cell simply replicates producing identical daughter cells every time. Examples of these and related reactions are given in the following figure. This reaction is known as the Pinacol rearrangement.
The vectors are made up of an origin of replication- This is a sequence of nucleotides from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted. There are a number of ways in which these recombinant DNAs are inserted into the host, namely – microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc. They can be conveniently manipulated as they are small enough and they are capable of carrying extra DNA which is weaved into them. The recombinant DNA technology emerged with the discovery of restriction enzymes in the year 1968 by Swiss microbiologist Werner Arber, Inserting the desired gene into the genome of the host is not as easy as it sounds. Draw the mechanism of its formation. The dehydration mechanism for a tertiary alcohol is analogous to that shown above for a secondary alcohol. Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have a very high copy number. Nitrogen fixation is carried out by cyanobacteria wherein desired genes can be used to enhance the productivity of crops and improvement of health. Plasmids are circular DNA molecules that are introduced from bacteria. Thus the recombinant DNA has to be introduced into the host. It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using restriction enzymes. They are not part of the main cellular genome. The technology used for producing artificial DNA through the combination of different genetic materials (DNA) from different sources is referred to as Recombinant DNA Technology. Note how the carbocation after the rearrangement is resonance stabilized by the oxygen.
The minor product being the same product as the one formed from the red arrows. It involves the selection of the desired gene for administration into the host followed by a selection of the perfect vector with which the gene has to be integrated and recombinant DNA formed. Clinical diagnosis – ELISA is an example where the application of recombinant. And at last, it has to be maintained in the host and carried forward to the offspring. Explain the roles of the following: (a) Restriction Enzymes. Gene cloning finds its applications in the agricultural field. For the example below, the trans diastereomer of the 2-butene product is most abundant. There are multiple steps, tools and other specific procedures followed in the recombinant DNA technology, which is used for producing artificial DNA to generate the desired product. Note: While the mechanism is instructive for the first part of the this answer. The restriction endonucleases are sequence-specific which are usually palindrome sequences and cut the DNA at specific points. The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i. e. free from other macromolecules. Applications Of Gene Cloning. Notice in the mechanism below that the alkene formed depends on which proton is abstracted: the red arrows show formation of the more substituted 2-butene, while the blue arrows show formation of the less substituted 1-butene.
The carbocation rearrangement would occur and determine the major and minor products as explained in the second part of this answer. However, the general idea behind each dehydration reaction is that the –OH group in the alcohol donates two electrons to H+ from the acid reagent, forming an alkyloxonium ion. Assume no rearrangement for the first two product mechanisms. Process of Recombinant DNA Technology. Secondary and tertiary alcohols dehydrate through the E1 mechanism. The predominance of the non-Zaitsev product (less substituted double bond) is presumed due to steric hindrance of the methylene group hydrogen atoms, which interferes with the approach of base at that site. Isolation of Genetic Material. The more substituted alkene is favored, as more substituted alkenes are relatively lower in energy. However, in this case the ion leaves first and forms a carbocation as the reaction intermediate. Host organism – into which the recombinant DNA is introduced. Primary alcohols undergo bimolecular elimination (E2 mechanism) while secondary and tertiary alcohols undergo unimolecular elimination (E1 mechanism). If the reaction is not sufficiently heated, the alcohols do not dehydrate to form alkenes, but react with one another to form ethers (e. g., the Williamson Ether Synthesis). Recall that according to Zaitsev's Rule, the more substituted alkenes are formed preferentially because they are more stable than less substituted alkenes. DNA cloning takes place through the insertion of DNA fragments into a tiny DNA molecule.
Draw an arrow pushing mechanism for the acid catalyzed dehydration of the following alcohol, make sure to draw both potential mechanisms. The hydroxyl oxygen donates two electrons to a proton from sulfuric acid (H2SO4), forming an alkyloxonium ion. Yeast cells, viruses, and Plasmids are the most commonly used vectors. Discuss the applications of recombination from the point of view of genetic engineering. Gene therapy in diseases like cancer, SCID etc.
Production of transgenic animals with improved quality of milk and egg. The lone pair of electrons on oxygen atom makes the –OH group weakly basic. Production of transgenic plants with improved qualities like insect and drought resistance and nutritional enrichment.
Additinally, trans alkenes are more stable than cis alkenes and are also the major product formed. They are two types, namely Endonucleases and Exonucleases. Similarly to the reaction above, secondary and tertiary –OH protonate to form alkyloxonium ions. Alcohols are amphoteric; they can act as both acid or base. The E2 elimination of 3º-alcohols under relatively non-acidic conditions may be accomplished by treatment with phosphorous oxychloride (POCl3) in pyridine.
The relative reactivity of alcohols in dehydration reactions is ranked as follows: Methanol < primary < secondary < tertiary. Recombinant DNA technology is widely used in Agriculture to produce genetically-modified organisms such as Flavr Savr tomatoes, golden rice rich in proteins, and Bt-cotton to protect the plant against ball worms and a lot more. Stay tuned with BYJU'S to learn more about the Recombinant DNA Technology, its tools, procedure and other related topics at BYJU'S Biology.
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