Recombinant DNA technology steps
- Recombination is defined as exchange of DNA sequence between different molecules, occurring either naturally or as a result of DNA manipulation. Recombinant DNA technique includes all the techniques involved in construction, study and use of recombinant DNA molecules.
- The recombinant DNA molecule is that DNA molecule which is created in test tube by ligating together, pieces of DNA that are not normally contiguous.
- Recombinant DNA involves breakage of a DNA molecule at desired places to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position.
- The product thus obtained is called recombinant DNA and the technique is often called genetic engineering. With this technique, genes can be isolated, cloned and characterized, so that the technique has led to significant progress in all areas of molecular biology.
- Using the recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. This became possible, because bacteria, phages and plasmids reproduce in their usual style, even insertion of foreign DNA, so that the inserted DNA also replicates faithfully with the parent DNA. This technique is called gene cloning.
Steps in Recombinant DNA Technology
Isolate DNA then cutting the DNA with restriction enzymes after that ligate into cloning vector then transform recombinant DNA molecule into host cell so that each transformed cell will divide many, many times to form a colony of millions of cells, each of which carries the recombinant DNA molecule (DNA clone).
- Isolation of Desired Gene
- Preparation of Vector
- Introduction of Recombinant DNA into Host cell
- Selection of recombinant host cell
- Expression of cloned gene
Isolation of desired DNA
For making recombinant organisms, a desired DNA fragment has to be introduced into the host cell, these desired DNAs can be obtain from the total genome of the cell either by restriction digestion (with restriction endonucleases or by mechanical shearing). The desired gene can be clone is located in the cell DNA of the source organisms along with several genes so firstly, it should be isolated from other genes of the cell DNA. For this purpose two methods are follows to isolate desired DNA from the genome of the cell. They are restriction digestion and mechanical shearing.
- Preheat 5ml CTAB (add 10μl mercaptoethanol to each 5ml CTAB) in a blue-topped 50ml centrifuge tube at 60-65°C. Remove and discard midribs and wrap laminae in aluminum foil and freeze in liquid nitrogen. 0.5 – 1.0 gm tissue/5ml CTAB (Can store leaf material after liquid Nitrogen – 1-2 days at –20°C or –80°C for longer periods)
- Gently crumble leaf tissue over a cold pestle of liquid nitrogen. Grind frozen leaves with one spatula of fine sand add 0.5 spatulae of PVPP powder after grinding.
- Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid leaving dry material around rim of tube. Adjust CTAB volume to give a slurry-like consistency, mix occasionally.
- Incubate for 60 min at 60°C
- Add equal volume of chloroform/iso-amyl alcohol (24:1), Mix for about 3min, then transfer contents to narrow bore centrifuge tubes. Balance by adding extra chlor/iso. Spin 5,000rpm for 10min (ensure correct tubes used), brake off. (For extra pure DNA isolation – spin and retain supernatant before chloroform extraction).
- Remove supernatant with wide-bore pastette (cut off blue tip) to clean tube, repeat chloroform extraction once. Supernatant should be clear, though may be coloured.
- Precipitate DNA with 0.66 vol. of cold isopropanol – can leave overnight. Spool out or spin down DNA, 2min at 2,000rpm.
- Transfer to 5ml wash buffer for 20min.
- Dry briefly and resuspend in 1ml T.E. (can be left overnight)
- Add 1μl 10mg/ml RNase to each 1ml T.E./DNA mixture and incubate for 60min at 37 °C. (If RNase in the sample doesn’t matter – stages 11 and 12 may be omitted)
- Dilute with 2 volumes TE and add 0.3vol 3M Sodium acetate (pH 8) +2.5 vol. cold 100% ethanol
- Spool DNA out. Air dry and resuspend in 0.5 to 1ml TE or water (takes time) and freeze until required.
Restriction digestion is the cutting of DNA into fragments by restriction enzymes.The total DNA of an organism is treated with restriction enzymes. These enzymes cut the cell DNA into many fragments; each fragment is differing in their size and molecular weight. These restriction enzymes cut the DNA into blunt end and cohesive end depending upon the mixture of these DNA fragments is electrophoresed on an agarose gel for a particular time By electrophoresis the mixture of these DNA fragments get separated on the basis of their size and molecular weight. Each DNA fragment obtained from the electrophoresis band is cloned into a suitable vector.
The restriction enzyme may cut the DNA at the middle of the desired gene and make it useless.
Genomic DNA is subjected to mechanical forces to cut genomic DNA into small fragments. Sonication with ultrasound cut this DNA randomly with the size of about 300-325 bp.
Each time it produces new DNA fragments This method cannot be applied for Eukaryotic organism because of presence of introns.
Recombinant DNA is a hybrid DNA formed by joining a desired foreign DNA and a vector DNA, It is indicated by rDNA. The vector DNA and genomic DNA fragments are mixed together. Cohesive end of the vector DNA anneals with genomic DNA fragment by complimentary base pairing and there is nick between these two DNA ends.
This nick is sealed by an enzyme called DNA ligase, It makes the Phosphodiester bond in between these two DNA fragments. Sometimes adapters, linkers and homopolymer tail are also used to join blunt ended DNA molecules.
The recombinant DNA can be introduced into the host cell by direct transformation and indirect transformation. In direct method, bacterial cell intake the recombinant DNAs in the medium like microinjection, liposomes, electroporation and particle bombardment method. In indirect method the pathogenic agents such as bacteriophage and Agrobacterium pick up the recombinant DNA and introduced it into plant cell.
DNA clone is a section of DNA that has been inserted into a vector molecule and then replicate in a host cell to form several copies.