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Cloning and Vector Chapter 3 Instructor : Prof. Myoung-Dong Kim T: 6458, [email protected] Room 411, Ag. Bld #3 Gene Cloning Cloning - a definition From the Greek - klon, a twig An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) An individual grown from a single somatic cell of its parent & genetically identical to it Clone: a collection of molecules or cells, all identical to an original molecule or cell DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest. Gene cloning When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a particular gene is copied (cloned) Why Clone DNA? A particular gene can be isolated and its nucleotide sequence determined Control sequences of DNA can be identified & analyzed Protein/enzyme/RNA function can be investigated Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc. Sources of DNA for Cloning 1) Chromosomal DNA 2) RNA converted to cDNA 3) PCR-amplified DNA PCR-amplified DNA Cloning Tools Restriction endonucleases Ligase Vectors Host Methods for introducing DNA into a host cell Cutting DNA Restriction endonucleases (restriction enzymes) • sticky ends • blunt ends Nomenclature • • • • • EcoRI E = genus (Escherichia) co = species (coli) R = strain I = # of enzyme Blunt & Sticky ends Pasting DNA Complementary ends (sticky ends) H-bond Ligase forms phosphodiester bond to seal strands together. Vectors Cloning vectors Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. 1 Plasmid vectors 2 Bacteriophage vectors 3 Cosmids 4 BACs & YACs Plasmid vectors Plasmid vectors are double-stranded, circular, selfreplicating, extra-chromosomal DNA molecules. Advantages: • Small, easy to handle • Straightforward selection strategies • Useful for cloning small DNA fragments (< 10kbp) Disadvantages: • Less useful for cloning large DNA fragments (> 10kbp) Plasmid vectors Plasmids are circular DNA molecules present in the cytoplasm of the bacteria Capable of autonomous replication Can transfer genes from one cell to other Act as vectors in genetic engineering. Can also present in Yeasts Plasmid vectors may encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules T.V.Rao MD Plasmid vector for cloning 1. Contains an origin of replication, allowing for replication independent of host’s genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site (MCS) 4. Easy to be isolated from the host cell. Plasmid vectors Bacteriophage vectors Advantages: • Useful for cloning large DNA fragments (10 - 23 kbp) • Inherent size selection for large inserts Disadvantages: • Less easy to handle l vectors Left arm: • head & tail proteins Right arm: • DNA synthesis • regulation • host lysis Deleted central region: • integration & excision • regulation Bacteriophage Cosmid vectors Combine the properties of plasmid vectors with the useful properties of the l cos site Advantages: • Useful for cloning very large DNA fragments (32 - 47 kbp) • Inherent size selection for large inserts • Handle like plasmids Disadvantages: • Not easy to handle very large plasmids • (~ 50 kbp) l ZAP BACs and YACs BACs : Bacterial Artificial Chromosomes YACs : Yeast Artificial Chromosomes Advantages: • Useful for cloning extremely large DNA fragments (100 - 2,000 kbp) • This is very important for genome sequencing projects Disadvantages: • Not easy to handle extremely large DNA molecules BAC vector oriS and oriE mediate replication parA and parB maintain single copy number ChloramphenicolR marker YAC vector large inserts ARS URA3 telomere centromere replication origin HIS3 markers telomere Capable of carrying inserts of 200 - 2000 kbp in yeast What determines the choice vector? insert size vector size restriction sites copy number cloning efficiency ability to screen for inserts what down-stream experiments do you plan? Expression vector Expression vector pSE420 • Polylinker: insert desired DNA • Amp resistance • trc promoter • lacO (operator) • Shine-Dalgarno (S/D) site (ribosome binding) • T1, T2 transcription terminators • lacI (lac repressor) growth inducer added cloned gene expressed; product produced Btech6 How to clone DNA • insertion of foreign DNA at BamHI site • tet resistance gene inactivated • transformants carrying foreign DNA are amp resistant but tetracycline sensitive transformation: transfer of genetic information via free DNA How to clone DNA Isolation of cloning vector (bacterial plasmid) & genesource DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest Screening of the clone The medium in this petri dish contains the antibiotic Kanamycin The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance Note the difference in growth Blue/White Color Screening lacZ lacZ functional enzyme X-gal product insert nonfunctional enzyme X-gal product Selecting Colonies with Recombinant Plasmids Colony hybridization DNA probe available? • part of same gene • orthologue from another species • synthetic oligonucleotide Bacteriophage lambda as a cloning vector transduction: transfer of host genes from one cell to another by a virus (Fig. 10.44, p. 311, Madigan et al.) Other methods for introducing DNA Electroporation: the use of an electric pulse to enable cells to take up DNA • Millisecond-length pulses open small pores in cell membranes • DNA can move into/out of the cells via pores cell plasmid donor microprojectile “gun” plasmid transformant desired transformant Transgenic plants may be produced with binary vector system in Agrobacterium tumefaciens (a) generalized plant cloning vector • ends of T-DNA (red) • ori (E. coli), ori (A. tumefaciens) • resistance markers (kan, spec) (b) can clone in E. coli; transfer to A. tumefaciens by conjugation (c) D-Ti = engineered Ti (to remove pathogenesis genes) (d) D-Ti will mobilize T-DNA of vector → plant cells grown in tissue culture (e) whole plants can be regenerated from recombinant cell