Lecture - 8 Gene transfer techniques: Vector mediated gene transfer, plasmids, cosmids, phages, YAC, BAC and HAC. Vectorless direct gene transfer. Cloning strategies
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> The desired gene sequence can be inserted into these vectors and then put back into yeast; the yeast machinery cannot differentiate between their own and foreign genes; hence the foreign gene is also replicated.
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Vectorless gene transfer:- When stable transformation is achieved by incorporating the desired gene into the genome of plant cells without the help of any biological factor, it is called direct gene transfer.
1. Partical gun method:-
• It is a physical method of gene transfer.
• It is also called Biolistic method.
• In this, tungsten or gold particles of 1-2µm diameter are coated with DNA and fired them into the cell by a particle gun.
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• Gold is expensive, but it is less harmful to cells.
• The cost of a particle gun is almost 15 lakhs.
• The particles are accelerated by compressed helium gas in the particle gun.
• DNA reaches inside the nucleus and becomes incorporated into the plant's genome.
• By this method, the gene is transferred to the meristem and embryo.
2. Liposome-mediated gene transfer:-
• Liposomes were discovered in the year 1960 by British haematologist “Dr.Alec D.Bangham”.
• These are the concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membrane lipid bilayer mainly composed of natural and synthetic phospholipids.
• Its Size range varies from 20nm - 3μm microns.
• Liposomes are spheres of lipids which can be used to transport the molecules into the cells.
• Liposomes deliver not only nucleic acids, but also other targeting ligands, like peptides, antibodies, aptamers, folic acid.
• Chemical modifications of liposome can efficiently improve their performance.
3. Chemical method:-
• It is also called as PEG mediated Gene Transfer.
• In this method gene transfer is induced by PEG. (PEG = Poly Ethylene Glycol)
• Transformation medium is added in a beaker that has a high concentration of Mg2 + ions.
• Now plant protoplasts are put into this nutritive medium.
• Now put the desired gene in the beaker.
• Now take the pH to 8.
• Now add 20% PEG.
• Now decrease the concentration of PEG and increase the concentration of Ca2+ ions.
• Now incubate for some time.
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• The transformation frequency increases 10 to 1000 times when plant protoplasts are transferred to ice after heat-shock at 45°C for 5 minutes just before adding DNA.
Introduction:-
> The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.
> It is popularly known as genetic engineering.
> Recombinat DNA:- When foreign gene is inserted into a vector, then it is called as recombinant DNA.
> Objective:- This is the natural mathod of amplification of gene of interest.
Process of Recombinant DNA Technology:- The complete process of recombinant DNA technology includes multiple steps-
Step-1. Isolation of Genetic Material:- The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.
Step-2.Cutting the gene at the recognition sites:- The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’.
Step-3. Ligation of DNA Molecules:- In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.
Step-4. Insertion of Recombinant DNA Into Host:- In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. Once the recombinant DNA is inserted into the host cell, it gets multiplied. As a result the inserted gene of interest is also multiplied.
Step-5. Amplifying the gene copies:- It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using restriction enzymes.
• Discovery:- The PCR technique was discovered by Kary Mullis in 1985.
• Thermocyclers are used to achieve different temperatures.
• PCR has three main steps -
i. Denaturation:- When dsDNA is heated, both its chains separate at a temperature of 90°C.
ii. Primer Annealing:- Primers are attached at the 5 'end of single chains at a temperature of 55°C.
iii. Polymerization:- DNA polymerase enzyme polymerize the primers at 70°C temperature.
• Heat stable DNA polymerase:-
Ø Normal DNA polymerase is heat sensitive. It is destroyed due to its deformation at 90°C temperature. Therefore, we cannot use normal DNA polymerase in PCR.
Ø Instead, we use heat stable DNA polymerase in PCR which can tolerate high temperature and does not deform.
