Selection and screening of recombinant clones
- 2. SELECTION – After the introduction of recombinant DNA into the host cells, it is essential to identify those cells which received rDNA molecule - screening (or) selection. – The vector or foreign DNA present in the recombinant cells expresses certain characters or traits, while non-recombinants do not expess the traits. – Following the methods for screening or selection of recombinant clones.
- 4. – The simplest example of Direct Selection occurs when the desired gene specifies resistance to an antibiotic. As an example will will consider an experiment to clone the gene for kanamcycin resistance from plasmid R6-5. – This plasmid carries genes for resistances to four antibiotics: kanamycin, chloramphenicol, streptomycin and sulphonamide. The kanamycin resistance gene lies within one of the 13 EcoRI fragments (a). – To clone this gene the EcoRI fragments of R6-5 would be inserted into the EcoRI site of a vector such as pBR322. The ligated mix will comprise many copies of 13 different recombinant DNA molecules, one set of which carries the gene for kanamycin resistance. – Insertional inactivation cannot be used to select recombinants when the EcoRI site of pBR322 is used. This is becuase this site does not lie in either the ampicillin or the tetracycline resistance of this plasmid. – But this is immaterial for cloning the kanamycin resistance gene because in this case the coloned gene can be used as the selectable marker. Transformants are plated onto kanamycin agar, on which the only cells able to survice and produce colonies are those recombinants that contain the cloned kanamycin resistance gene(c).
- 7. – Insertional inactivation is the inactivation of a gene upon insertion of another gene in its place or within its coding sequence. – This helps in selection of recombinant colonies in rDNA technology.
- 10. – It is a powerful method for screening recombinants. – In this method a reporter gene lacZ is inserted in the vector (encodes β-galactosidase). – β-galactosidase breaks a synthetic substrate, X-gal into an insoluble blue colured product. – If a foreign gene is inserted into lacZ, this gene will be inactivated; therefore no blue colour will develop. – The host cells containing recombinant will form white coloured substrate on the medium containing X-gal. – The host cells containing non recombinants wil turn blue in colour. – On the basis of colony colour the recombinants can be selected.
- 13. – Colony blot hybridization is applied to DNA or RNA released from blotted microbial colonies. – The microbial colonies are transferred (blotted) to a membrane. – The cells are lysed in place to release the nucleic acids. – The RNA or DNA (after denaturation) is fixed to the filter and hybridized with a labelled probe. – Blocking reagent may be added prior to the probe to prevent unspecific binding. – Excess probe is washed away and the membrane is visualized by UV or autoradiography. – Colony blot hybridization can be used for screening clones or bacterial isolates.
- 15. – Instead of radio-labelling of DNA molecules, antibodies are used to identify the colonies developed that synthesize antigens encoded by the foreign DNA present in plasmids of the bacterial clones. – 1) replica plating – 2) lysis of cells using chloroform vapour/ high temp. – 3) Making gentle contack with a solid support ( cellulose filter paper). – 4) detection of antigen antibody complex by incubating the cellulose filter paper with a radio labellled second antibody. – 5) the antibodies which do not react are washed off. – 6) the determination of antigen antibody complex is determined by passing through x ray
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