1 Recombinant DNA technology

2 Recombinant DNA technologyMethods 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:

3 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

4 Hydrolysis of DNA by restriction endonucleasesFIGURE 13.4 Hydrolysis of DNA by restriction endonucleases. (a) Separation of ends. (b) Resealing of ends by DNA ligase. Fig. 13-4, p.333

5 Production of recombinant DNAFIGURE 13.5 (1) Foreign DNA sequences can be inserted into plasmid vectors by opening the circular plasmid with a restriction endonuclease. (2) The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. Fig. 13-5, p.334

6 Restriction Endonucleases (r.e)Enzymes produced by bacteria that hydrolyze the phosphodiester backbone of DNA at specific sequences 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.

7 Table 13-1, p.335

8 Methylation of endogenous DNA protects it from cleavage by its own restriction endonucleases.

9 Dolly In July, 2016, four identical clones of Dolly (Daisy, Debbie, Dianna and Denise) were alive and healthy at nine years old. The late Dolly, the most famous sheep in the world, produced by cloning techniques. p.331

10 The cloning process that produced Dolly

11 The cloning of a virus Fig. 13-6, p.336FIGURE 13.6 The cloning of a virus. The progeny of each individual phage (bacterial virus) infects and destroys bacteria on the petri dish, leaving clear spots known as plaques. Each plaque indicates the presence of a clone. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-6, p.336

12 The cloning of cells Fig. 13-7, p.336FIGURE 13.7 The cloning of cells. Each individual cell divides many times, producing a colony of progeny. Each colony is a clone. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-7, p.336

13 The cloning of human DNA fragments with a viral vectorFIGURE 13.8 The cloning of human DNA fragments with a viral vector. Human DNA is inserted into viral DNA and then cloned. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-8, p.336

14 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. DNA plasmids—extrachromosomal selfreplicating genetic elements of a bacterial cell. p.338

15 For A Successful ExperimentWhen 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.

16 A vector cloning site containing multiple restriction sitesFIGURE A vector cloning site containing multiple restriction sites. This is called a polylinker or multiple cloning site (MCS). The colored amino acids are from the lacZ gene that is part of the plasmid. The MCS does not disrupt the normal reading frame of this sequence, so this plasmid can be used for blue/white screening (see text). (Adapted from Figure , in Ausubel, F. M., et al., 1987, Current Protocols in Molecular Biology. New York: John Wiley and Sons. Used by permission.) Fig , p.339

17 Cloning with pU plasmidsFIGURE The pUC series of plasmids is very popular. They have extensive multiple cloning sites. Here we see an example of directional cloning. Two different restriction enzymes are used to cut open the MCS and to cut out a piece of DNA to be cloned. As a result, the DNA that is to be inserted can be incorporated in only one orientation. Fig , p.340

18 Cloning It refers to creating a genetically identical populationDNA can be combined by using r.e that create sticky ends in the DNA. This rDNA has a target DNA sequence of interest The target DNA sequence is carried in some type of vector, usually a bacterial plasmid or a virus 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 Organisms that are carrying the target DNA are identified through a process called selection, which often involves antibiotic resistance.

19 Synthesis of insulin in humansFIGURE Synthesis of insulin in humans. The insulin gene is a split gene. The intervening sequence (intron) encodes an RNA transcript that is spliced out of the mRNA. Only the portions of the gene called exons are reflected in the base sequence of mRNA. Once protein synthesis takes place, the polypeptide is folded, cut, and spliced. The end product, active insulin, has two polypeptide chains as a result. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig , p.345

20 Production of recombinant human insulinFIGURE Active human insulin can be produced in bacteria by the use of two separate batches of E. coli. Each batch produces one of the two chains, the A chain or the B chain. The two chains are mixed to produce active insulin. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig , p.346

21 Electroporation. Foreign DNA can be introduced into plant cells by electroporation, the application of intense electric fields to make their plasma membranes transiently permeable.

22 Genetic Engineering (g.e)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.

23 Genetic Engineering in AgricultureDisease 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. Increased milk production giving cows bovine somatotropin (BST) “growth hormone”…. Good predator attraction straw berry gene on mustard plants produces a chemical attractant for predator mites that eat the herbivorous spider mites.

24 Transgenic tomato plant : Recombinant DNA methods have produced plants that resist defoliation by caterpillars, with longer shelf life. FIGURE A transgenic tomato plant. Recombinant DNA methods have produced plants that resist defoliation by caterpillars. Tomatoes with a longer shelf life are another result of this research. Fig , p.349

25 Steps of making recombinant DNAIsolation 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).

26 1. Isolation of nucleic acidsIs 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).

27 DNA extraction from human uses blood sample as a source of DNADNA 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.

28 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.

29 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(non-complementary) 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.

30 sticky ends

31 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 blunt-ends of DNA chains to convert them into sticky ends.

32 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.

33 4. Amplification of recombinant DNAI. 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.

34 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.

35 Molecular cloning is based on two basic principles1.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.

36 Selecting for recombinant DNA in a bacterial plasmidFIGURE 13.9 Selecting for recombinant DNA in a bacterial plasmid. The plasmid also contains a gene for antibiotic resistance. When bacteria are grown in a medium that contains the antibiotic, those that have acquired a plasmid will grow. Bacteria without a plasmid cannot grow in this medium. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig. 13-9, p.338

37 Specific steps of cloningA. 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.

38 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.

39 Plasmid pBR322 FIGURE One of the first widely used cloning vectors, the plasmid pBR322. This 4363-basepair plasmid contains an origin of replication (ori) and genes encoding resistance to the drugs ampicillin (ampr) and tetracycline (tet r). The locations of restriction endonuclease cleavage sites are indicated. Fig , p.339

40 B. Generation and Ligation of Inserts into VectorThe 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.

41

42 DNA Library It is a collection of clones of an entire genomeThe genome is digested with r.e & the pieces are cloned into vectors & transformed into cell lines 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 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.

43

44 Formation of cDNA Fig. 13-21, p.352FIGURE Reverse transcriptase catalyzes the synthesis of a strand of complementary DNA (cDNA) on a template of mRNA. The cDNA directs the synthesis of a second strand, which is then incorporated into a vector. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig , p.352

45 Steps involved in the construction of a DNA libraryFIGURE Steps involved in the construction of a DNA library. All the DNA of a given organism is extracted and treated with a restriction enzyme. The DNA fragments are incorporated into bacterial plasmids. Specific clones can be selected. The remaining clones are saved for future use. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig , p.350

46 C. Choice of an E. coli hostUsually 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.

47 D. Transformation of E. coli with Recombinant VectorTransformation 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

48 population Clone selection via blue/white screeningInclusion of ampicillin in the agar allows only bacteria with a plasmid to grow, because these plasmids provide antibiotic resistance population FIGURE Clone selection via blue/white screening. The pUC plasmid contains a gene for ampicillin resistance and the lacZ gene. The latter produces the -subunit of -galactosidase. Transformed cells are plated on an agar medium containing ampicillin and a dye called X-gal. The lacZ gene is found inside the multiple cloning site of the plasmid. When the plasmid and the DNA to be cloned are cut with a restriction enzyme and then mixed together, two possibilities result. The DNA insert can be incorporated as shown (red insert seen inside plasmid), or the plasmid can recircularize without the insert. When this mixture is used to transform bacteria, there can be three products. Left side: The bacteria take up a plasmid that has the insert. This plasmid confers ampicillin resistance to the cells, but the lacZ gene is inactivated by the presence of the insert. These cells grow and are the normal off-white color of bacterial colonies. Middle: The bacteria take up the recircularized plasmid. This plasmid confers ampicillin resistance, so the cells grow. The plasmid makes the -subunit of -galactosidase. The -galactosidase cleaves the X-gal, causing the dye to turn blue, so these cells grow with a blue color. Right side: Bacteria take up no plasmid at all. These cells do not grow, due to their sensitivity to ampicillin. (Adapted with permission from Dealing with Genes: The Language of Heredity, by Paul Berg and Maxine Singer, © 1992 by University Science Books.) Fig , p.341

49 Three types of colonies are produced:1. Transformed bacteria containing recombinant plasmid 2. Transformed bacteria containing non-recombinant plasmid 3. Non-transformed bacteria

50 Identification of cells containing plasmidsInclusion of X-gal allows for blue-white colony screening. Recombinant clones will have white colonies while non-recombinant will have blue colonies.