PCR is Polymerase Chain Reaction. It is used to amplify the DNA that is make multiple copies of the desired DNA 'in vitro' using DNA polymerase and two sets of primers.
Three main steps of PCR are-
1. Denaturation- where the DNA strands are heated so that the two strands separate and form single strands to which primer can attach.
2. Primer annealing - in this step the primer attach to the complementary strands on the single stranded DNA.
3. Extension of primers- DNA polymerase extends the primers and forms double stranded DNA.
The three steps of PCR:
2. Primer annealing
3. Extension of primers.
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.
List the features required in vectors that facilitate cloning . With a suitable diagram show the E.coli cloning vector with restriction sites.
Features required in vectors that facilitate cloning are :
1. Origin of replication (Ori) which helps in autonomous replication and also controls the copy number.
2. Selectable marker which helps in isolation of transformed cell.
3. Cloning sites which provide sites at which foreign DNA can be inserted . These sites also help in selection of recombinants.
4. Vectors like Agrobacterium tumefaciens ( in plants) and retroviruses (in animals) can be made non pathogenic and be used to fascilitate cloning.
Fig. 7.4. E. coli Cloning Vector pBR322 showing restriction sites (Hindlll, EcoRI, BamHI, Sal I, Pvu II, Pst I, Cla l), oriV and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.
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.
Genetic engineering is a technique for artificially and deliberately modifying DNA (genes) to suit human needs. It includes techniques like creating recombinant , gene cloning and gene transfer.
Steps in genetic engineering:
1. Identification and isolation of genetic material.
2. Cutting of the DNA at specific locations using restriction enzymes.
3. Amplification of the desired gene using Polymerase Chain reaction.
4. Introduction of gene of interest into host cell or organism.
5.The gene is cloned and the desired gene product the recombinant protein is obtained.
6. Downstream processing is done which includes processing like and separation purification
Fig. Diagram showing Various steps involved in DNA recombinant technology for the production of a recombinant protein.