L21. techniques for selection, screening and characterization of transformants
- 1. Techniques for Selection, Screening and Characterization of Transformants 1 Lecture- 21
- 2. Selection of Transformants • In recombinant DNA technology, after introduction of recombinant DNA molecules into host cells, it is important to select the host cell that takes up the DNA construct (transformed cell) from those that do not • It can be done by selectable marker genes or reporter genes 2
- 3. Selectable Marker Genes • Selectable marker genes are present on vector into which the DNA of interest has been cloned. These genes protect the organism from a selective agent that would normally kill it. • All cells that do not contain the foreign DNA are killed in the presence of selective agent and only the desired ones are left behind Commonly used selectable marker genes are: Neomysin phosphotransferase - npt III Glyphosate oxidoreductase – gox Hypoxanthine-guanine phosphoribosyltransferase – hprt Thymidine kinase - tk 3
- 4. Reporter Genes An alternative to selectable marker gene is a reporter gene which helps in distinguishing between wanted and unwanted cells. Ideally a reporter gene should have following properties: i. Its product should be easy to assay ii. There should be little or no endogenous activity for this gene iii. It should be non-toxic iv. It should tolerate N-terminal fusions Commonly used reporter genes are: Chloramphenicol acetyltransferase β-galactosidase Nopaline synthase Octopine synthase 4
- 5. Identification of clones of interest • Once the transformants are selected, the next step is to identify a specific transformant containing the insert of desired gene • This can be done by three methods Sequence-dependent screening Protein structure/function-dependent screening Complementation method 5
- 6. Sequence-dependent screening • Sequence-dependent screening can be achieved by exploiting the homologous sequence of the desired gene • Sufficient information about the sequence of interest is required to make suitable probes and primers • This is done by following two methods: • Nucleic acid hybridization technique • PCR 6
- 7. 7 • Nucleic acid hybridization technique The clones (colonies or plaques) of a library are transferred to a membrane made up of nitrocellulose or nylon. The colonies on the membrane are gently lysed and DNA coming out of the cells is immobilized on the membrane. The desired clone is selected by hybridization of the immobilized DNA with a labeled DNA probe. • PCR technique The clones are screened by colony PCR method by using primers which are designed based on the available sequence information.
- 8. Protein structure/function-dependent screening • Besides nucleic acid sequences, the structure/function of its expressed product can also be analyzed • Screening the product of a clone applies only to expression libraries • The clone can be identified because its product is recognized by an antibody or ligand or because the biological activity of the protein is preserved and can be assayed 8
- 9. 9 •For this method the genomic DNA or cDNA library should be constructed in an expression vector. The desired clone is identified on the basis of the structure/function of the protein produced. •The protein product is separated by lysing the cells of clones and subjecting the cell lysates to polyacrylamide gel electrophoresis. The desired protein on the gel is identified with the help of molecular weight markers. •To confirm the identified protein, the protein band on the gel is transferred to a nitrocellulose or polyvinylidene membrane. This technique is called western blotting. The desired protein product on the membrane can be confirmed by binding it to a specific antibody/ligand.
- 10. 10 Complementation method • All the clones of a library are pooled and recombinant vector molecules are isolated from the pooled cells. A mutant of the organism is obtained in which the gene of interest is mutated. The mutated cells are transformed with the recombinant vector molecules isolated above. • The mutated cells in which restoration of the wild type function occurs are selected. Recombinant vector molecules isolated from these cells contain the wild type copy of the gene of interest. In some cases the library can be directly conjugated with the mutant cells. • The bacterial genes involved in biosynthesis of amino acids, nucleotide bases and vitamins can be easily cloned by this method. The nodulation genes of Rhizobium were cloned by this approach. • The complementation method can be used only for those genes for which the restored function can be easily selected.
- 11. Nucleic Acid Blotting and Hybridization • Membranes used • Nitocellulose membrane • Nylon membrane • Polyvinylidene difluoride 11
- 12. An Overview of Nucleic Acid Hybridization 12
- 13. Transfer of Nucleic Acid from Gel to Membrane • Upward capillary transfer • Downward capillary transfer • Simultaneous transfer to two membranes • Electrophoretic transfer • Vacuum transfer 13
- 14. Capilary transfer of nucleic acids from gel to membrane; (a) upward capilary transfer, (b) downward capilary transfer, (c) simultaneous transfer of two membranes 14
- 16. Methods of Immobilization • After the transfer of nucleic acid from gel to membrane or directly spotting on the membrane following two methods of immobilization are used for fixation of DNA or RNA on membranes Baking Cross-linking by UV irradiation 16