RECOMBINANT DNA technology
1. This is a modern biotechnological advance , in which a desired gene fragment can be inserted in to a cloning vector and the resulting DNA (Recombinant DNA) can be amplified in suitable host. 2. A vector can be a plasmid, cosmid,bacterophage,retroviruses, animal and plant viruses or artificial chromosomes like YAC, BAC,or HAC.(Yeast artificial chromosome, bacterial........) 3. The rec. DNA produced can be amplified or cloned in a suitable vector like bacteria for plamids, cosmids or bacterophages, plant and animal cells for viruses . Involves five steps:
1. Enzyme restriction digest of DNA sample.
2. Enzyme restriction digest of DNA plasmid vector (same Res.Enzyme). 3. Ligation of DNA sample products and plasmid vector.
4. Transformation with the ligation products.
5. Growth on agar plates with selection for antibiotic resistance. [pic]
← APPLICATION OF RECOMBINANT DNA technology
← Gene therapy
← Recombinant Vaccines
← Genetically modified crops
← Monoclonal antibodies
← Cell/tissue culture
← Production of next generation antibiotics
← Bioterrorism detection
DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube. Cloning vectors share four common properties: 1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning. Main types of cloning vectors
Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), HAC, retrovirus, baculovirus vector etc.
← Bacterial cells may contain extra-chromosomal DNA called plasmids. ← Plasmids are usually represented by small, circular DNA. ← Some plasmids are present in multiple copies in the cell ← F –plasmid= capable of conjugation
← R-plamid= Encode antibiotic resistance
← Plasmid vectors are ≈1.2–3kb and can clone up to 12 kb of insert DNA IT CONTAINS,
1. replication origin (ORI) sequence : DNA segment recognized by the cellular DNA-replication enzymes. 2. a gene that permits selection, antibiotic resistance like ampr;tetr etc ampr encodes the enzyme beta-lactamase, which inactivates ampicillin. Selective marker is required for maintenance of plasmid in the cell. 3. Multiple cloning site(mcs) :Exogenous DNA can be inserted in mcs .It is a DNA segment with several unique sites for restriction endo- nucleases located next to each other.Restriction sites of the polylinker are not present anywhere else in the plasmid.Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector. 4. Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb).
5. ColE1 based, pUC vehicles commercially available ones, eg pGEM3, pBlueScript
6. Some are expression vectors and have sequences that allow RNA polymerase to transcribe genes (promoters, Terminators and ribosome binding sites)
7. DNA sequencing primers
CLONING IN PLASMID VECTOR
← Examples of plasmid vectors
← First artificial cloning vector developed in 1977from E.Coli (By Boliver and Rodrigues). ← Contain 4361 base pairs
← Two selectable markers (Ampicillin and Tetracycline resistance genes) ← Several unique restriction sites scattered throughout plasmid 0 restriction sites for Hinlll,Eco RV,BamH1 etc. ← Some retriction sites lie within antibiotic resistance genes = means of screening for inserts ← ColE1 ORIGIN(Ori)