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Recombinant DNA and
Genetic Engineering
Chapter 8
Purpose: Co-opt bacteria in order to grow
large quantities of a particular
DNA sequence (e.g., a gene).
Why is this important?
Ex: Human genome = 3 x 109 bp
One gene = 103 to 104 bp, or
~0.0003% of total genomic DNA
Clone = Genetically identical individuals.
Examples: Bacteria cells in a single colony.
Many cells
Colony = pile of bacteria cells in one spot
on petri dish, all derived from one
cell following many rounds of cell
division -- Genetically identical.
DNA clone = many identical copies of a single
DNA molecule.
Example: A DNA molecule present in the bacteria cells
in a single colony.
Many cells
o
o
o = plasmid DNA
o
o
o
o
o
o
o
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o
o
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o
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By extracting plasmid DNA from colony of bacteria
cells, one can obtain useable quantities of a
DNA with identical sequence.
Restriction Enzymes
Enzymes produced by bacteria.
Purpose: primitive immune system to fight infection by bacteriophage.
Bind to specific DNA sequences & make cuts in DNA backbone.
Example: EcoRI - Binds to & cuts
GAATTC
CTTAAG
Modification Enzymes: Protect bacteria cell’s own DNA from
its own restriction enzymes.
Example: EcoRI Methylase - Adds methyl groups to middle A’s
of EcoRI recognition sequence
m
GAATTC
CTTAAG
m
Restriction Enzymes
3’
5’
5’
3’
5’
3’
5’
3’
Many restriction enzymes’ recognition sites are palindromic.
Those that make staggered nicks, produce restriction fragments with
“sticky ends”.
Sticky ends = short complementary single strands.
Making Recombinant DNA Molecules
1. Cut DNA you want to
clone & vector DNA with
restriction enzyme to
create complementary
sticky ends.
2. Mix DNAs together & add
DNA ligase.
3. Complementary sticky
ends base pair, but DNA
ligase must seal nick to
permanently join the DNAs
together.
Forms phosphodiester
bonds