ge Long Answer Type @ jestions 1. Explain briefly the various processes of recombinant DNA technology. — (May 2014) (March 2014) A. Recombinant DNA technology involves several steps in spacific sequence such as a) Isolation of DNA b) Fragmentation of DNA by restriction endonucleases c) Isolation of a desired DNA fragment a) Ligation of the DNA fragment into a vector e) Transferring the recombinant DNA into the host f) Culturing the host cells in amedium at large scale g) Extraction of the desired product. a) Isolationof DNA: / A DNA is enclosed within the membranes. To release DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids, bacterial cells / plants or animal tissue are treated with enzymes such as lysozyme (bacteria) cellulose (plant cells), chitinase (fungus) to digest cell wall. ii) RNA can be removed by treatment with ribonuclease. iii) Proteins can be removed by treatment with protease. iv) Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. b) Fragmentation of DNA by restriction endonucleases : Cutting of DNA at specific locations can be done by using restriction enzymes. The purified DNA is incubated with the specific restriction enzymes at conditions optimum for the enzyme to act. ¢) Isolation of a desired DNA fragment : Is carried out using agarose gel electrophoresis the DNA is negatively charged, it moves towards the positive electrode or anode and in the proces, DNA separates out. The desired DNA fragment is eluted out. d) Amplification of the gene of interest : Using Polymerase Chain Reaction (PCR) is @ ~reaction in which amplification of specific DNA sequences is carried out in vitro. i) PCR technique require: . a) A DNA template which is a double-stranded DNA that needs to be amplified. b)Primers are small chemically made oligonucleotides of about 10-18 nucleotides that are complementary to a region of template DNA. -) Enzymes used are taq polymerase (from Thermus aquaticus) and the vent polymerase (from Thermococcus litoralis).ii) Steps in PCR: a) Denaturation of double - stranded DNA is carried out by applying high temperature of 95°C for 15 seconds. Each separated single-stranded strand acts as a template for DNA synthesis. b) Anneling is carried out in two sets of primers. Which are added and anneal to the 3' end of each separated strand. Primers act as initiator of replication. c) Extension is done by DNA polymerase of primers by adding nucleotides complementary to the template provided in the reaction. d)A thermostable DNA polymerase (taq polymerase) is used in the reaction which can tolerate the high temperature of the reaction. e) These steps are repeated many times to obtain several copies of desired DNA. d) Ligation of the DNA fragment into a vector requires a vector DNA and source DNA. i) These are cut with the same endonuclease to obtain sticky ends. ii) Both are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form recombinant DNA. Foreign DNA DOCOOCOOCOOOCK ‘Same restriction enzyme cutting both foreign DNA and vector DNA at specific point 3QC 30 DCOOKK Ligases join foreign DNA to plasmid Transformation cot EON f Cells divide Diagrammatic representation of recombinant DNA technologye) Insertion of recombinant DNA into the host cell : Organism occurs by several methods, before which the recipient cells are made competent to receive the DNA. i) If recombinant DNA carrying antibiotic resistance (eg & ampicillin) is transferred into E.coli cells, the host cell is transformed into ampicillin resistant cells ii) The ampicillin resistant gene can be called as selectable marker. iii) When transformed cells are grown on agar plates containing ampicillin, only transformants will grow and other will die. f) — Culturing the host cells is carried out in appropriate medium at optimal conditions, The DNA gets multiplied and express itself to form desired products. Extraction of desired gene products is carried out by following steps. i) Aprotein encoded gene expressed in a heterologous host is called recombinant protein, ii) Cells having genes of interest can be grown on a small scale or on a large scale. iii) In small scale cells are grown on cultures and then expressed protein is extracted and purified by various separation methods. iv) In large scale cells are grown in a continuous culture system in which fresh medium is added from one side to maintain cells growth phase and the desired protein is collected from the other side. 2. Give a brief account of the tools of recombinant DNA technology. A. The tools of recombinant DNA technology i) Restriction enzymes ii) Polymerase enzymes iii) Ligases iv) Vectors v) Host organism i) Restriction enzymes : Belong to a larger class or enzymes called Nucleases. These are two kinds. a) Exonucleases : Exonucleases remove nucleotides from the ends of the DNA. b) Endonucleases : Endonucleases make cuts as specific positions with in the DNA. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases.on the opposite strands. Eg : EcoRI recognises 5' GAATTC 3' sites on the DNA and cuts in betweenGandA. C23" c T TA A G i Naming of Restriction Enzymes : > The first letter of the name comes from the genus and the next two lett name of the species of the prokaryotic cell from which it is isolated“) The next letter comes from the strain of the prokaryote. » The Roman numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium. Eg: EcoRI from Escherichia coli RY13 Hind I is from Haemophilus influenzae Bam HI is from Bacillus amyloliquefaciens ii) Polymerase enzymes : Thermas aquaticus a bacterium yields DNA polymerase used in biotechnology. i) This enzyme remains qctive during the high temperature applied during denaturation of double-stranded DNA. ii) It extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. iii) Repeated amplification is achieved by this enzyme. The amplified fragments, if desired can be used to ligate with a vector for further cloning. iii) Ligases : The enzyme DNA ligase joins the complementary ends of the plasmid DNA with that of desired gene by covalent bonding to regenerate a circular hybrid called Recombinant (r) DNA or chimeric DNA. ; iv) Vectors : The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host called vector. Vectors used for multiplying the foreign DNA sequence are called cloning vectors. Commonly used vectors are plasmids, bacteriophages, cosmids. Properties of cloning vector 1) It must have low molecular weight. 2) It must have unique cleavage site for the activity of restriction enzymes at single point. 3) It must be able to replicate inside the host cell after its introduction (through ori gene - origin of replication). : 4) The vectors requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of transformants. v). Host organisms : Competent host for transformation with Recombinant DNA is required as tool.2. Give a brief account of the tools of recombinant DNA technology. Ans. Key tools are : 1) Restriction enzymes : ‘Two enzymes responsible for restricting the growth of Bacteriophage in Escherichia coli were isolated in the year 1963. One of these added methy! groups to DNA and the other cut DNA. The latter was called restriction endonuclease, The first restriction endonuclease - Hind II which cut DNA molecules at a particular point by recognising a specific sequence of six base pairs, called recognition sequence for Hind II. Today, more than 900 restriction enzymes were isolated from over 200 Strains of Bacteria, each of which recognises a different recognition sequence. E CORI is a restriction enzyme in which, the first letter comes from the genus (Escherichia), and the second two letters from the species of the Prokaryotic cell [coli], the letter 'R is derived from the name of strain. Roman numbers indicate the order in which the enzymes were isolated from that strain of Bacteria. Restriction enzymes belong to a larger class of enzymes called nucleases. They are of two types. a) Exonucleases which remove nucleotides from the ends of the DNA. b) Endonucleases which make cuts at specific location with in the DNA. — 7 ESMost restriction enzymes cut the two strands of DNA double helix at different locations. such a cleavage is know as staggered cut. E CoRI recognises 5' GAATT 3' sites on the DNA snd cuts it between G and A results in the formation of sticky ends or cohesive end pieces. this stickyness of the ends facilitates the action of enzyme DNA ligase. Cloning vectors : The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vector. Vectors used for multiplying the foreign DNA sequences are called cloning vectors. Commonly used cloning vectors are plasmids, bacteriophages, cosmids. Plasmids are extrachromosomal circular DNA molecules present in almost all bacterial species. They are inheritable and carry few genes are easy to isolate snd reintroduce into the bacterium (Host). Features required to facilitate cloning into a vector : a) Origin of replication : (ori) This is a sequence from where replication starts and any piece of DNA when linked to this. sequence can be made to replicate within host cells. It is also responsible for controlling the copy number of the linked DNA. b) Selectable marker : In addition to ‘ori, the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the any transformants normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin etc., are useful selectable markers for E.Coli. c) Cloning sites : In order to link the alien DNA, the vector needs to have very few, preferably single recognition sites for the restriction enzymes. d) Molecular weight : The cloning vector should have low molecular weight. e) Vectors for cloning genes in plants and animals : The tumour inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector such that it is ae thogenic to plants. Similarly retroviruses have also been disarmed and are now desirable genes into animal cells, used to delive