Transcript Lecture 18 POWERPOINT here

Genetic Engineering 1
Lecture 18
Pages 323 - 340
The Tools of Molecular
Biology
Old fashioned way was to breed for what you wanted
10_01_experiment.DNA .jpg
Man has been doing this since the Agricultural revolution,
some 10,000 years ago… The Chinese have been doing it!
Mendel did it too!
What do we do NOW…
• Breeding is still used.
• However, quicker and more results orientated
methods now exist due to technology.
• First, we may need to filter the cell types…
• Label target cells with florescent dye attached
to characteristic cell specific proteins
– Brain cells have some unique proteins
– Liver cells have other unique proteins, etc.
Still used - an automated cell sorter
10_02_cell_sorter.jpg
Once you have the cells what
next?
• OPTIONS
• Issue was to examine the DNA in a consistent
manner
• Best method (Restriction Digestion) is to use
restriction enzymes (REs)
– Come mainly from bacteria
• They are bacterial immune systems against foreign DNA
– They are all Proteins
– Many types have been isolated from different
species of bacteria
– Scientists use REs individually or in a mix
10_04_Restrict.nuclease.jpg
What do you do with these
digested fragments of DNA?
• Isolate those that you want to work on
• How?
– Best method is to use gel electrophoresis
• Agarose
• Polyacrylamide
10_05_gel.electrophor.jpg
Then what do you do with this
piece of DNA?
• EITHER
– Clone
– Sequence
• Rely on the use of dideoxy nucleotides
10_07_1_enzym.dideoxy.jpg
10_07_2_enzym.dideoxy.jpg
10_08_DNA.sequencing .jpg
Other methods used
Shotgun cloning
In the Dark…
10_09_Shotgun.sequenc.jpg
Use computer
software to assemble
Problems with this method
What can they be?
10_10_Repetit.sequence.jpg
YACs
BACs
Cloning Techniques used to obtain
DNA sequence information
10_11_BAC.clones.jpg
10_12_de_renaturation.jpg
10_13_hybridization.jpg
Blotting
Purpose = to make a permanent record of
the results of a gel electrophoresis.
The compass • Southern blots - DNA
• Northern blots - RNA
• Western blots - Proteins
• Eastern blots - ?
10_14_1_Southrn.blotting.jpg
10_14_2_Southrn.blotting.jpg
DNA microassays
New techniques
Many uses
DNA Chips
10_15_DNA.microarrays .jpg