Transcript IntrotoBiotechRestrictionEnzymes2011 (1)

Biotechnology
• various tools in biotechnology have made it possible for humans to
manipulate and control DNA sequences.
• recombinant DNA is a fragment of DNA which contains sequences that
come from at least 2 different sources. (ie. A gene for human insulin located in
a bacterial DNA).
CSI
Restriction Enzymes (Endonucleases)
• Enzymes that are able to cut double stranded DNA at specific sequences.
• They originate from bacteria and are used in their native environment to destroy
(by chopping up) any DNA that is not property of the bacteria.
• Restriction enzymes will cut DNA at a specific sequence (called a recognition
site).
• One example, EcoRI, cuts DNA at the following sequence.
• Most recognition sites (as above) are 4-8 base pairs long and are palindromic (read
the same bases 5’-3’ on one strand as they do 5’-3’ on the other strand).
• EcoRI will scan along a DNA strand until it finds it’s recognition site and then it
breaks the phosphodiester bond between G and A, resulting in the formation of two
DNA strands instead of one.
Creepy Restriction Enzyme Movie
• some restriction enzymes (like EcoRI) produce cuts in the DNA that result in the
formation of sticky ends on the DNA fragments that are formed.
• sticky ends indicates that unpaired bases are left hanging off the cut.
other restriction enzymes produce blunt ends, that is, the DNA is cut directly down
the middle.
• cuts that produce sticky ends are more preferable to a biotechnologist because
they can be joined to other sticky end fragments with the help of H-bonding between
complimentary bases.
Plasmid Mapping
Using restriction enzymes – it is possible to create a map of a plasmid that
will enable the insertion of a known gene at a particular location – this is
called molecular cloning.