Ability to replicate independently (so that a lot of copies could be generated) A recognition...
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Transcript of Ability to replicate independently (so that a lot of copies could be generated) A recognition...
• 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
• Vectors– Generally plasmids or viruses
• Plasmids– Small circular DNA molecule– Introduced into bacteria by transformation
• 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
•Transform E.coli with plasmid
–Treat with CaCl2
–Makes walls more permeable to small DNA molecules–Can also be introduced by electroporation
• 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
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 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
• http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=003&n=84&i=168&v=category&o=|26|132|131.1|37|14|60|72|84|&ns=209&uid=0&rau=0
DNA of interest can also be isolated by PCR amplification