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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 Polymerases DNA Polymerase – catalyzes the polymerization of deoxyribonucleotides along the template strand DNA-dependent RNA Polymerase Restriction Endonucleases Nucleases 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 ++ T4 DNA ligase for “blunt” ends 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 tocertain antibiotics carry one or more genes some of which confer resistance tocertain 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 plasmid replication: stringent or relaxed plasmid replication: stringent or relaxed

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 viruses that infect bacteria viruses that infect bacteria known dsDNA sequence of ~ 50 kb known dsDNA sequence of ~ 50 kb linear double-stranded molecule with single-stranded complementary ends linear double-stranded molecule with single-stranded complementary ends cohesive termini (cos region) cohesive termini (cos region)

13 can accept large pieces of foreign DNA can accept large pieces of foreign DNA tremendous improvement over the years tremendous improvement over the years can be reconstituted in vitro can be reconstituted in vitro Desirable properties of λ phage:

14 modified plasmids containing cos sequences modified plasmids containing cos sequences carry an ORI & an antibiotic resistance marker carry an ORI & an antibiotic resistance marker can accommodate ~35 to 45 kb of foreign DNA can accommodate ~35 to 45 kb of foreign DNA can be propagated as plasmids can be propagated as plasmids can be introduced into host by standard procedures can be introduced into host by standard procedures chief technical problems occur when used for library construction chief technical problems occur when used for library construction

15  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:

16  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

17 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)

18 Transfection of the DNA DNA is packaged in vitro into phage particles DNA is packaged in vitro into phage particles phages are allowed to infect bacterial cells phages are allowed to infect bacterial cells term also used in DNA transfer to eukaryotic cells term also used in DNA transfer to eukaryotic cells DNA is transiently expressed DNA is transiently expressedConjugation natural transmission from donor to recipient natural transmission from donor to recipient host cell that is not readily transformed host cell that is not readily transformed form cell to cell junctions form cell to cell junctions

19  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 plaque formation plaque formation

20 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 plaque type plaque type blue-white selection (β-galactosidase) blue-white selection (β-galactosidase) specific (hybridization, antibodies, PCR) specific (hybridization, antibodies, PCR)

21 LacZ gene – encodes for β-galactosidase LacZ gene – encodes for β-galactosidase X-Gal – substrate for the enzyme X-Gal – substrate for the enzyme IPTG (isopropyl-[beta]-D-thiogalactopyranoside)– inducer IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer cloning sites within the LacZ gene cloning sites within the LacZ gene disruption of gene by insertion disruption of gene by insertion of the foreign DNA blue – functional protein blue – functional protein white – non-functional protein white – non-functional protein

22  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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25 /cloningvectors.html


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