Download 042310_recombinant_DNA2

Characterstics of a vector
• 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)
Recombinant DNA Technology
• Vectors
– Generally plasmids or viruses
• Plasmids
– Small circular DNA molecule
– Introduced into bacteria by transformation
Characterstics of a vector
• 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)
•Transform E.coli with
plasmid
–Treat with CaCl2
–Makes walls more
permeable to small
DNA molecules
–Can also be
introduced by
electroporation
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.
Recombinant DNA Technology
Second challenge is solved with the help of lacZ
gene.
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 betagalactoside bond
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 colonies of 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 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
• Gels:
– Are porous
– Agarose (a polysaccharide from red algae)
• Use electrophoresis to separate
molecules on the basis of:
– Size and electrical charge
Eco RI
Eco RI
+
Size
stds
Cut vector
Insert DNA
DNA of interest can also be
isolated by PCR amplification
DNA of interest can also be
isolated by PCR amplification
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