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Recombinant DNA technology Recombinant DNA technology • Methods used to join together (recombine) different DNA segments that are not found together in nature. • This technique is used in genetic analysis to serve several applications: 1. In Vitro Mutagenesis It is much easier to make mutation in isolated gene than when it is part of a complex organism structure 2. Study of the gene properties (genotype) 3. Study the expression of gene product (phenotype) 4. Produce large quantity of medically or agriculturally or industrially important gene product e.g Insulin production in large quantity by bacteria Hydrolysis of DNA by restriction endonucleases Fig. 13-4, p.333 Production of recombinant DNA Fig. 13-5, p.334 Restriction Endonucleases (r.e) 1. Enzymes produced by bacteria that hydrolyze the phosphodiester backbone of DNA at specific sequences 2. The sequences targeted by r.e are palindromes, meaning their sequence reads the same on both strands going in the same direction 3. Most r.e cut DNA in a way that leaves sticky ends that are very useful for recombining DNA from different sources. Table 13-1, p.335 p.335 Dolly 1996-2003 In July, 2016, four identical clones of Dolly (Daisy, Debbie, Dianna and Denise) were alive and healthy at nine years old. p.331 The cloning process that produced Dolly The cloning of a virus Fig. 13-6, p.336 The cloning of cells Fig. 13-7, p.336 The cloning of human DNA fragments with a viral vector Fig. 13-8, p.336 DNA Plasmid Extra chromosomal self-replicating genetic elements of a bacterial cell & can be transferred from one strain of a bacterial species to another by cell-to-cell contact. p.338 For A Successful Experiment When bacteria take up a plasmid, we say they have been transformed Bacteria are encouraged to take up foreign DNA by: 1.heat-shock the bacteria at 42 C. followed by placing them on ice. 2. Place them in an electric field “electroporation” Then selection through selectable markers on the plasmid. A vector cloning site containing multiple restriction sites Fig. 13-11, p.339 Cloning with pU plasmids Fig. 13-12, p.340 Cloning 1. It refers to creating a genetically identical population 2. DNA can be combined by using r.e that create sticky ends in the DNA. This rDNA has a target DNA sequence of interest 3. The target DNA sequence is carried in some type of vector, usually a bacterial plasmid or a virus 4. The target DNA sequence is inserted into a host organism & the natural doubling time of the organism is used to create many copies of the target DNA sequence 5. Organisms that are carrying the target DNA are identified through a process called selection, which often involves antibiotic resistance. Synthesis of insulin in humans Fig. 13-15, p.345 Production of recombinant human insulin Fig. 13-16, p.346 Electroporation. Foreign DNA can be introduced into plant cells by electroporation, the application of intense electric fields to make their plasma membranes transiently permeable. Genetic Engineering (g.e) 1. It is the process of inserting genes of interest into specific organisms for either a medical or scientific benefit 2. Gene therapy is the process of inserting a missing gene into an organism 3. Bacteria are often used as the factories to produce a protein from a cloned gene. This has led to the production of human insulin & erythropoietin..etc. 4. The gene must be cloned into an expression vector, usually a plasmid with special features that allows it to be transcribed & translated in a host cell. Genetic Engineering in Agriculture 1. 2. 3. 4. Disease resistance e.g. corn & cotton Nitrogen fixation Frost-free plants e.g strawberries & potatoes Tomatoes with a long shef life deactivating the gene in tomato which produce ethylene. 5. Increased milk production giving cows bovine somatotropin (BST) “growth hormone”…. 6. Good predator attraction straw berry gene on mustard plants produces a chemical attractant for predator mites that eat the herbivorous spider mites. Transgenic tomato plant : Recombinant DNA methods have produced plants that resist defoliation by caterpillars, with longer shelf life. Fig. 13-18, p.349 Steps of making recombinant DNA • Isolation of DNA Cutting DNA in to small pieces with restriction enzymes Ligate the pieces into cloning vector Transform recombinant DNA molecule into host cell The transformed cell divides many times to form a colony of millions of cells, each carries the recombinant DNA molecule (DNA clone). 1. Isolation of nucleic acids Is the separation of DNA free from other major molecules such as RNA and proteins, lipids and polysaccharides. The isolation procedure mainly involve: DNA isolation from bacteria involves lysis of cell wall by a lysozyme enzyme then alkali denaturation treatment followed by solvents extraction (phenol/chloroform/isoamyl alcohol mixture ) which separates chromosomal DNA from plasmid DNA (remains circular shape and not affected by alkali treatment). • DNA extraction from human uses blood sample as a source of DNA. In particular, the DNA of white blood cells is isolated in a procedure almost similar to the bacterial DNA without the need for the use of lysozyme enzyme. 2. Cutting DNA • DNA can be cut into large fragments by Restriction enzymes (r.e). • They are group of endonucleases found as protective enzymes in bacteria to destroy foreign DNA in a process called restriction. The host bacterial DNA itself is methylated by a modification enzyme (a methylase) to be protected from the restriction enzyme’s activity. Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes. The product of restriction enzymes cutting to the DNA is either a sticky (complementary) or blunt(noncomplementary) two ends. In sticky ends: the terminal part of DNA are unequal cohesive single strands which can easily combined together due to complementary property between them. sticky ends However , because sticky ends are usually more needed in recombinant DNA technology than blunt end , an enzyme called Terminal deoxynucleotidyl transferase can be used to add nucleotides to the bluntends of DNA chains to convert them into sticky ends. 3. Joining of hybrid DNA By cutting a donor DNA and receiver DNA with the same restriction enzyme, the ends of the two DNA will have the same sticky ends. These two DNAs are mixed in a tube to rejoin together due to the matching of their sticky ends. A small gap will be left that can be sealed by ligase. 4. Amplification of recombinant DNA I. Cloning of recombinant DNA It is the method of replicating recombinant DNA inside living cell to generate large population of cells containing identical copies of this type of DNA. The objective of cloning is to replicate recombinant DNA in large amounts, so that it can be used for genetic analysis. • Since the chemical structure of DNA fundamentally the same in all living organisms, any segment of foreign DNA from an organism is inserted into host DNA of living organism capable of replication, then the foreign DNA will be replicated along with the host cell's DNA during cell division. Molecular cloning is based on two basic principles 1.DNA fragments are inserted into plasmid vectors to produce recombinant DNA 2. The insert-vector recombinant molecules are transported into living cells, usually E.coli, which is grown up in colonies to make copies of the recombinant DNA. Selecting for recombinant DNA in a bacterial plasmid Fig. 13-9, p.338 Specific steps of cloning A. The selection of a vector B. Generation of foreign DNA fragments (containing particular gene) and ligate the foreign DNA into vector to make insert-vector recombinant. C. The selection of appropriate (competent) E. coli strain D. Transforming competent E. coli with recombinant vector E. Grow E. coli colonies to replicate recombinant vectors and select colonies containing the recombinants DNA. F. Characterizing the properties of DNA inserts by genetic analysis. A. Cloning Vectors Cloning vector is a plasmid that can be modified to carry new genes, which must have: • An origin of replication. • A selectable marker (antibiotic resistance gene, such as ampicillin resistance( ampr) or tetracycline resistance( tetr). • Multiple cloning site (MCS) a site where insertion of foreign DNA will not disrupt replication or inactivate essential markers. Plasmid pBR322 Fig. 13-10, p.339 B. Generation and Ligation of Inserts into Vector The standard procedure for creating the recombinant molecule involves cleaving the DNA of interest [the insert] and the vector with the same restriction enzyme, followed by incubation with DNA ligase to ligate the insert into the vector. DNA Library 1. It is a collection of clones of an entire genome 2. The genome is digested with r.e & the pieces are cloned into vectors & transformed into cell lines 3. Specific radioactive probes to a sequence of interest are reacted to filters that have copies of the bacterial colonies in the library. The probe binds to the sequence of interest, and the colony’s location can be seen by autoradiography 4. A cDNA library is constructed by using reverse transcriptase to make DNA from mRNA in a cell. This cDNA is then used to construct a library similar to a genomic DNA library. Formation of cDNA Fig. 13-21, p.352 Steps involved in the construction of a DNA library Fig. 13-19, p.350 C. Choice of an E. coli host • Usually an E. coli mutant called lacZΔM15 is used as a host for the recombinant vector. This mutant is characterized by having inactive β-galactosidase activity due to deletion in the N-terminal part of the enzyme protein coded by defective lacZ gene. • In the mean time , the defective part of this lacZ gene has been inserted into the vector(plasmid DNA).So, when the plasmid vector is transferred the bacterial host the combined parts complement to each others to give the active enzyme. A test for the formation of active enzyme can be detected by converting a blue colored X-gal dye into white color. D. Transformation of E. coli with Recombinant Vector • Transformation is the process of making the bacteria to take up the recombinant vector molecule. Nucleic acids do not enter bacteria under their own power but usually they require certain procedure involving treating the bacteria with ice-cold solutions of CaCl2 followed by short heating to 42 Clone selection via Inclusion of ampicillin in the agar allows only blue/white screening bacteria with a plasmid to grow, because these plasmids provide antibiotic resistance Fig. 13-13, p.341 Three types of colonies are produced: 1. Transformed bacteria containing recombinant plasmid 2. Transformed bacteria containing nonrecombinant plasmid 3. Non-transformed bacteria Identification of cells containing plasmids Inclusion of X-gal allows for blue-white colony screening. Recombinant clones will have white colonies while non-recombinant will have blue colonies.