(ii) Three steps of PCR
2. Primer annealing and
3. Extension of primers.
â² Fig. 7.2. The three steps of PCR
2. Insertion of the isolated gene in a suitable vector.
3. Introduction of vector into a suitable organism / host cell.
4. Selection of transformed host cells.
5. Multiplication or cloning of the introduced gene in the host organism.
1. The bacterial cells are made competent to take up recombinant DNA by treating them with a specific concentration of calcium, that increases the efficiency with which DNA enters the cell through the pores in its cell wall.
2. Microinjection is a method in which the recombinant DNA is directly injected into the nucleus-of the animal cell with the help of mirco-needles or micropipettes.
3. Gene gun or biolistics is a method suitable for plant cells, where plant cells are bombarded with high-velocity microparticles of gold or tungsten coated with DNA.
4. Disarmed pathogens can be allowed to infect the host cell, which then transfer the recombinant DNA into the host.
5. Electroporation in which electricity is applied to create transient pores in the host cell so that it takes up Recombinant DNA with ease.
What is genetic engineering? Explain briefly the distinct steps common to all genetic engineering technology.
With the help of diagrams show the different steps in the formation of recombinant DNA by action of restriction endonuclease.
Steps in genetic engineering:
1. Identification and isolation of agronomically important genes.
2. Cloning of isolate genes in a vector.
3. Introduction of gene into plaint protoplast cell / tissues with the use of gene transfer method.
4. Culture and regeneration complete plant on suitable selection medium.
5. Integration of foreign gene in the transgenic plants by using molecular techniques.
â² Fig. 7.1. Diagram showing Various steps involved in DNA recombinant technology for the production of a recombinant protein.
List the features required in vectors that facilitate cloning . With a suitable diagram show the E.coli cloning vector with restriction sites.
1. Origin of replication (Ori)
2. Selectable marker
3. Cloning sites
4. Vectors for cloning genes in plants and animals Sites of cloning vector
â² Fig. 7.4. E. coli Cloning Vector pBR322 showing restriction sites (Hindlll, EcoRI, BamHI, Sal I, Pvu II, Pst I, Clal), oriV and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.