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Technique involving the insertion of a fragment of foreign DNA into a vector capable of replicating autonomously in a host cell (usually Escherichia coli). Growing the host cell allows the production of multiple copies of the inserted DNA for use in a variety of purposes. Foreign DNA Host organism Vector DNA for cloning Means of inserting foreign DNA into the vector Method of placing the in vitro modified DNA into the host cell Methods for selecting and/or screening cells that carry the inserted foreign DNA Restriction Endonucleases Polymerases DNA Polymerase – catalyzes the polymerization of deoxyribonucleotides along the template strand DNA-dependent RNA Polymerase Nucleases Enzymes capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids Other Modifying Enzymes Ligases forms phosphodiester bonds to join two pieces of DNA utilizes ATP in the presence of Mg++ T4 DNA ligase for “blunt” ends Kinases transfers phosphate groups from donor molecules phosphorylase Phosphatases catalyzes the removal of 5’-phosphate residues Foreign DNA PCR product genomic DNA complementary DNA (cDNA) Host organism bacterial host – E. coli eukaryotic host – yeast (Saccharomyces cerevisiae) other hosts – other yeasts, insect cells, etc. Vector DNA DNA molecule that functions as a “molecular carrier” that carry the DNA of interest into the host cell & facilitates its replication. Plasmids – used in cloning small segments of DNA (10-15 kb) Bacteriophage λ – used in cloning larger segments of DNA (~20 kb) Cosmids – plasmids containing DNA sequences (cos) from bacteriophage λ used to clone larger fragments of up to 45 Kb • small circular dsDNA that autonomously replicates apart from the chromosome of the host cell • “molecular parasites” • carry one or more genes some of which confer resistance to certain antibiotics • origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host • plasmid replication: stringent or relaxed Desirable properties of plasmids: small size known DNA sequence high copy number a selectable marker a second selectable gene large number of unique restriction sites http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif • viruses that infect bacteria • known dsDNA sequence of ~ 50 kb • linear double-stranded molecule with single-stranded complementary ends • cohesive termini (cos region) http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif Desirable properties of λ phage: • can accept large pieces of foreign DNA • tremendous improvement over the years • can be reconstituted in vitro • modified plasmids containing cos sequences • carry an ORI & an antibiotic resistance marker • can accommodate ~35 to 45 kb of foreign DNA • can be propagated as plasmids • can be introduced into host by standard procedures • chief technical problems occur when used for library construction Means of inserting foreign DNA into the vector Ligation of the DNA into the linearized vector http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif Requirements for a ligation reaction: • two or more fragments of DNA (blunt/cohesive) • buffer containing ATP • T4 DNA ligase Method of placing the in vitro modified DNA into the host cell Transformation into the host cell • bacterial cells take up naked DNA molecules • cells are made “competent” • cells treated with ice-cold CaCl2 then heat-shocked • efficiency of 107 to 108 transformed colonies/μg DNA • maximum transformation frequency of 10-3 • “electric field-mediated membrane permeabilization” • high strength electric field in the presence of DNA • protocols differ for various species • efficiencies of 109 per μg DNA (3 kb) & 106 (136 kb) http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg Electroporation of the DNA into the host cell Conjugation • natural transmission from donor to recipient • host cell that is not readily transformed • form cell to cell junctions Transfection of the DNA • DNA is packaged in vitro into phage particles • phages are allowed to infect bacterial cells • term also used in DNA transfer to eukaryotic cells • DNA is transiently expressed Methods for selecting and/or screening cells that carry the inserted foreign DNA Selection refers to application of conditions that favors the growth of cells or phages that carry the vector or vector and insert. • antibiotic resistance • nutrient requirements • plaque formation Screening allows all cells to grow, but tests the resulting clones for the presence of the insert in the vector. • antibiotic resistance/sensitivity • nutrient requirements • plaque type • blue-white selection (β-galactosidase) • specific (hybridization, antibodies, PCR) • LacZ gene – encodes for β-galactosidase • X-Gal – substrate for the enzyme • IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer • cloning sites within the LacZ gene • disruption of gene by insertion of the foreign DNA • blue – functional protein • white – non-functional protein http://www.eppendorfna.com/applications/images/gel_cleanup1.jpg DNA isolation for: making probes restriction mapping sequencing reintroduction into organism Establishment of collections: DNA Libraries Further molecular studies: production of special proteins ? http://www.bio.indiana.edu/~chenlab/potocols/MolecularClonging.htm http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html http://www.protocol-online.org/forums/index.php?showforum=30 http://bioresearch.ac.uk/browse/mesh/D003001.html http://www.jax.org/~jcs/techniques/techniques.html http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/clones.html http://opbs.okstate.edu/~Melcher/MG/MGW4/MG428.html http://www-micro.msb.le.ac.uk/109/GeneticEngineering.html http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer /cloningvectors.html