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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.

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Presentation on theme: "Technique involving the insertion of a fragment of foreign DNA into a vector capable of replicating autonomously in a host cell (usually Escherichia coli."— Presentation transcript:

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2 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.

3  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

4 Nucleases Polymerases DNA Polymerase – catalyzes the polymerization of deoxyribonucleotides along the template strand DNA-dependent RNA Polymerase Restriction Endonucleases Enzymes capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids

5 Other Modifying Enzymes Ligases forms phosphodiester bonds to join two pieces of DNA utilizes ATP in the presence of Mg ++ Kinases transfers phosphate groups from donor molecules phosphorylasePhosphatases catalyzes the removal of 5’-phosphate residues

6  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. other hosts – other yeasts, insect cells, etc.

7  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) Cosmids – plasmids containing DNA sequences ( cos ) from bacteriophage λ used to clone larger fragments of up to 45 Kb Bacteriophage λ – used in cloning larger segments of DNA (~20 kb)

8 small circular dsDNA that autonomously replicates apart from the chromosome of the host cell small circular dsDNA that autonomously replicates apart from the chromosome of the host cell “molecular parasites” “molecular parasites” carry one or more genes some of which confer resistance to certain antibiotics 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 origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host

9 small size small size known DNA sequence known DNA sequence high copy number high copy number a selectable marker a selectable marker a second selectable gene a second selectable gene large number of unique restriction sites large number of unique restriction sites Desirable properties of plasmids:

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12  Means of inserting foreign DNA into the vector Ligation of the DNA into the linearized vector two or more fragments of DNA (blunt/cohesive) two or more fragments of DNA (blunt/cohesive) buffer containing ATP buffer containing ATP T4 DNA ligase T4 DNA ligase Requirements for a ligation reaction:

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14  Method of placing the in vitro modified DNA into the host cell Transformation into the host cell bacterial cells take up naked DNA molecules bacterial cells take up naked DNA molecules cells are made “competent” cells are made “competent” cells treated with ice-cold CaCl 2 then heat-shocked cells treated with ice-cold CaCl 2 then heat-shocked efficiency of 10 7 to 10 8 transformed colonies/μg DNA efficiency of 10 7 to 10 8 transformed colonies/μg DNA maximum transformation frequency of maximum transformation frequency of 10 -3

15 Electroporation of the DNA into the host cell “electric field-mediated membrane permeabilization” “electric field-mediated membrane permeabilization” high strength electric field in the presence of DNA high strength electric field in the presence of DNA protocols differ for various species protocols differ for various species efficiencies of 10 9 per μg DNA (3 kb) & 10 6 (136 kb) efficiencies of 10 9 per μg DNA (3 kb) & 10 6 (136 kb)

16  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 antibiotic resistance nutrient requirements nutrient requirements

17 Screening allows all cells to grow, but tests the resulting clones for the presence of the insert in the vector. antibiotic resistance/sensitivity antibiotic resistance/sensitivity nutrient requirements nutrient requirements blue-white selection (β-galactosidase) blue-white selection (β-galactosidase) specific (hybridization, antibodies, PCR) specific (hybridization, antibodies, PCR)

18 Positive selection disruption of the lacZα-ccdB gene permits growth of positive recombinants

19  DNA isolation for: making probes restriction mapping sequencing reintroduction into organism  Establishment of collections: DNA Libraries  Further molecular studies: production of special proteins

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21 /cloningvectors.html


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