Biotechnology : Principles and Processes
Tools of Recombinant DNA Technology
- Three types of tools used in r – DNA Technology are Enzymes, Vectors, host.
- Enzymes are of 2 types – Lysing enzymes [that breaks open the cell] and cleaving enzymes [that breaks the DNA].
- Lysing enzymes – Eg. Lysozyme, cellulose, chitinases etc.,
- Cleaving enzymes are of 3 types – Exonuclease (remove DNA from terminal ends), Endonuclease (removes DNA within itself, but act only on single strand) and Restriction endonuclease (Cuts The DNA duplex), there by it produces sticky and blunt ends.
- Restriction Endonulceases (R.E) are of three types , I, II and III, out of which types II is widely accepted and used in Laboratory due to its specificity in cutting the recognised base pairs of the DNA and its simplicity in requiring only Mg2+ ions.
- Type I and III requires ATP, Mg2+ and S-Adenosyl methionine for its activity and also they are less specific in action.
- Type II R.E → Eg. E Co R I acts on image _________ and cuts in between G and A, there by it produces sticky ends.
E → Escherichia (Genus)
Co → Coli (Species)
R → RY 13 (Strain / sub species)
I → First enzyme isolation from it (E Coli RY 13)
- Hind II was the first discovered R.E.
- All the R.E has RM [Restriction / Modification (or) Methylation] system to protect the organism from which it is isolated. [discovered by Wemer and Arber].
- Source and vector DNA has to be cleaved by the same R.E.
- R.E acts only on palindromic DNA sequence.
- Cleaved DNA fragments are separated according to the size of the fragments using Agarose Gel Electrophoresis (AGE), by using electric field and a staining dye called Ethidium Bromide (Et Br).
- DNA is negatively charged and so it moves towards the applied positive field.
- Other enzymes used is cloning
⇒ DNA Ligase → Molecular glues
⇒ Alkaline phosphatase → removes phosphate from 5′ end of DNA/ RNA
⇒ Reverse Transcriptase → To synthesize DNA from RNA
⇒ DNA Polymerase → to synthesize DNA from DNA
- The smallest DNA fragmet moves far in the Agarose gel after electrophoresis and the largest DNA stays at the top because of its higher molecular weight.
- Basically vectors / vehicle / carriers are of 2 types – plasmid and Bacteriophage.
- The first artificially constructed vector was PBR322
P → Plasmid
BR → Boliver and Rodriguez
322 → Unique identification number.
- A vector should prossess a selectable marker, ORI (origin of replication) and cloning sites, where a selectable marker is to differentiate transformants and Non – transformants ; ORI is to start the replication process and cloning sites to insert the transgene into the vector.
- Selectable markers are usually Antibiotic resistance genes like tetracycline, Ampicillin resistance genes, through which transformants are identified by the technique called Insertional inactivation method / Blue white screening method, where Blue colonies represent non – transformants and white colonies represent transformants.
- Bacteriophage vectors are of 2 types – Lambda and MI3. [where Lambda phage has 48 kb and is double stranded ; M13 is of 6 kb and is single stranded] and they are advantageous as they have larger insert capacity and easy detection.
- Other vectors are cosmid, BAC/YAC, Phagemid, Viral vectors and shuttle vectors.
- Gene can be inserted into a competent host using vector (or) without a vector. [direct gene transfer (or) vectors less gene transfer]. Like microinjection, Electroporation, chemical mediated gene transfer (PEG - Poly Ethylene Glycol) and Biolistics/gene gun (or) Bioblaster method (Tungsten micro projectiles).
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