Gene cloning presentation
- 2. Gene Cloning: The insertion of a fragment of DNA carrying a gene into a cloning vector and subsequent propagation of recombinant DNA molecules into many copies is known as gene cloning. Gene Cloning:
- 3. •Gene cloning involves using bacteria to make multiple copies of a gene •Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell •Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA •This results in the production of multiple copies of a single gene
- 4. Gene Manipulation: Gene manipulation is defined as the formation of a new combination of a heritable material by the insertion of nucleic acid molecules (DNA molecules) into bacterial plasmid or any other vector so as to allow their incorporation in to the host organism, in which they are capable of containing propagation.
- 5. Figure 20.2 Bacterium Bacterial chromosome Plasmid 2 1 3 4 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvestedCopies of gene Basic research and various applications Basic research on protein Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth
- 8. •Transformation: • A bacterium takes up DNA from the medium. • Recombination takes place between introduced genes and the bacterial chromosome. Gene Transfer in Bacteria
- 9. Transformation •Methods: •Electroporation •Freeze Thaw Method •Calcium Chloride Mediated Transformation •Calcium Phosphate Mediated Transformation
- 10. Calcium Chloride Method •Treatment with calcium chloride in the early log phase of growth for Competence •Bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions Chloride Ions E.Coli
- 11. Calcium Chloride Method • As the chloride ions enter the cell, water molecules accompany the charged particle • Influx of water causes the cells to swell and is necessary for the uptake of DNA • The exact mechanism of this uptake is unknown.
- 12. Calcium Chloride Method • DNA of interest is then added to the cells
- 13. Calcium Chloride Method •Calcium chloride treatment be followed by addition of DNA of interest then by heat. •The heat shock step is necessary for the uptake of DNA.
- 14. Calcium Chloride Method •Temperatures > 42degC: Bacteria’s ability to uptake DNA reduces •Extreme temperatures: Bacteria dies.
- 15. Calcium Chloride Method •After the heat shock step intact plasmid DNA molecules replicate in bacterial host cells •To help the bacterial cells recover from the heat shock cells are briefly incubated with non- selective growth media
- 16. Calcium Chloride Method • As the cells recover, plasmid genes are expressed • Bacterial colonies selected using antibiotic selection techniques
- 18. screening technique Lose of antibiotic resistance. Colony hybridization. Immuno chemical method.
- 19. PCR Cloning Method PCR cloning differs from traditional cloning in that the DNA fragment of interest, and even the vector, can be amplified by the Polymerase Chain Reaction (PCR) and ligated together, without the use of restriction enzymes. PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts. Typically, a PCR reaction is performed to amplify the sequence of interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to transformation. Early PCR cloning often used Taq DNA Polymerase to amplify the gene. This results in a PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase. These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation.
- 21. High-fidelity DNA polymerases are also now routinely used to amplify sequences with the PCR product containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a typical ligation reaction or by the action of an "activated" vector that contains a covalently attached enzyme, typically Topoisomerse I, which facilitates the vector:insert joining. Some PCR cloning systems contain engineered "suicide" vectors that include a toxic gene into which the PCR product must be successfully ligated to allow propagation of the strain that takes up the recombinant molecule during transformation. A typical drawback common to many PCR cloning methods is a dedicated vector that must be used. These vectors are typically sold by suppliers, like NEB, in a ready-to-use linearized format and can add significant expense to the total cost of cloning. Also, the use of specific vectors restricts the researcher's choice of antibiotic resistance, promoter identity, fusion partners, and other regulatory elements.
- 22. Advantages: High efficiency, with dedicated vectors Amenable to high throughput Disadvantages: Limited vector choices Higher cost Lack of sequence control at junction Multi-fragment cloning is not straight forward Directional cloning is difficult
- 23. Thank you
Editor's Notes
- #6: Figure 20.2 A preview of gene cloning and some uses of cloned genes.