1. Recombinant DNA and Genetic Engineering
Chapter 8

2. 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.

3. 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.

4. DNA clone = many identical copies of a single
DNA molecule.
Example: A DNA molecule present in the bacteria cells
in a single colony.

5. By extracting plasmid DNA from colony of bacteria
Many cells o
o = plasmid DNA o o o o o o o o o o o o o o o
By extracting plasmid DNA from colony of bacteria
cells, one can obtain useable quantities of a
DNA with identical sequence.

6. 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
GAATTC
CTTAAG m

7. Restriction Enzymes 3’ 5’ 3’ 5’ 3’ 5’ 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.

8. Making Recombinant DNA Molecules
Cut DNA you want to clone & vector DNA with restriction enzyme to create complementary sticky ends.
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