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Ability to replicate independently ( so that a lot of copies could be generated) A recognition sequence for a restriction enzyme ( so that we can introduce our DNA of interest ) Reporter genes ( to confirm we have successfully introduced the vector into the host cell )

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slide1
Ability to replicate independently (so that a lot of copies could be generated)

A recognition sequence for a restriction enzyme (so that we can introduce our DNA of interest)

Reporter genes (to confirm we have successfully introduced the vector into the host cell)

Small size in comparison with host’s chromosomes (for easy manipulation)

Characterstics of a vector

recombinant dna technology
Recombinant DNA Technology
  • Vectors
    • Generally plasmids or viruses
  • Plasmids
    • Small circular DNA molecule
    • Introduced into bacteria by transformation
slide4
Small size in comparison with host’s chromosomes (for easy manipulation)

Ability to replicate independently (so that a lot of copies could be generated)

A recognition sequence for a restriction enzyme (so that we can introduce our DNA of interest)

Reporter genes (to confirm we have successfully introduced the vector into the host cell)

Characterstics of a vector

slide6

Transform E.coli with plasmid

    • Treat with CaCl2
    • Makes walls more permeable to small DNA molecules
    • Can also be introduced by electroporation
recombinant dna technology1
Recombinant DNA Technology
  • Two challenges remain:

First, how can you make sure that all the bacteria that is growing contain a plasmid?

Second, how can you identify which of the bacteria contains the recombinant plasmid.

  • Bacterial plasmids carry two reporter genes to overcome these challenges.
  • First problem is solved with the help of antibiotic (ampicillin) resistance on the plasmid vector.
slide9

Recombinant DNA Technology

Second challenge is solved with the help of lacZ gene.

recombinant dna technology2
Recombinant DNA Technology

Lac Z gene

  • Genetic indicator system
    • From lac operon
    • Codes for enzyme beta-galactosidase
    • Cleaves beta-galactoside bonds
  • Cleaves a synthetic beta-galactoside
    • 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal)
    • Galactose with a blue dye attached by a beta-galactoside bond
recombinant dna technology3
Recombinant DNA Technology

X-gal (galactose + blue dye) is colorless

  • If the beta-galactoside bond in X-gal is cleaved after taken up by the bacteria:
    • The dye is released from X-gal
    • Results in blue coloniesof bacteria
  • Why?
    • The lac Z is not interrupted
    • Beta-galactosidase is produced
    • X-gal is cleaved releasing the dye
    • The colonies are blue
recombinant dna technology4
Recombinant DNA Technology

X-gal (galactose + blue dye) is colorless

  • If the lac Z gene is interrupted with a foreign DNA
    • The gene is inactivated (the beta-galactosidase is inactive)
    • The dye is not released
    • The colonies are white
  • Final confirmation is obtained by retrieving the plasmid DNA from E. coli cells and performing restriction digestion to examine cloned DNA
viewing dna fragments
Viewing DNA Fragments
  • Gels:
    • Are porous
    • Agarose (a polysaccharide from red algae)
  • Use electrophoresis to separate molecules on the basis of:
    • Size and electrical charge
slide18

Eco RI

Eco RI

+

Size

stds

Cut vector

Insert DNA

dna of interest can also be isolated by pcr amplification1
DNA of interest can also be isolated by PCR amplification
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