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RECOMBINANT DNA TECHNOLOGY (THE NEEDED ITEMS (3) DNA cloning vector, 1)…
RECOMBINANT DNA TECHNOLOGY
rDNA ?
- cut and paste DNA from other sources = DNA cloning
- Clone = a cutting ( twig) used to propagate a plant , ex. orchid.
- Cloning = reproduction of whole organism without sexual fertilization between gametes.
- Gene cloning = insertion of foreign DNA (gene) into another organism
THE NEEDED ITEMS
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Important features
- able to replicate autonomously in bacterial host
- has a site for insertion of foreign DNA
- has a selectable antibiotic resistant marker
- pieces of DNA that accept, carry and
replicate (clone) other pieces of DNA
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- Initially originated from bacteria (closed circular double stranded DNA present in E.coli
- enzyme that joining two DNA fragments ( between 2 compatible sticky ends) - synthesize phosphodiester bond
common features :
- 1) Did not discriminate the source of DNA ;
RE can cut any DNA restriction site and
DNA ligase can joint together an DNA`s from any two sources
- 1) Restriction endonuclease (RE)
- Cutting is digestion
- Found in bacteria, used as defense against virus infection
- Shows specificity by cutting the specific sq on DNA (restriction site)
- RE cuts double stranded DNA by breaking phosphate=sugar
- RE cutting creates
- 1) single stranded overhanging ends of DNA
( sticky 5ends and sticky 3 ends)
- 2) double stranded ends (blunt ends)
e.g = EcoRI, BamHI, Sau3A
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- screening of recombinants
- Usage of selectable markers available on DNA plasmid :
- 1) Antibiotic resistant genes
- 2) lac Z gene (blue/white colony screening)
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