recombinant dna technology. further historical perspective geneticists have known for a long time...
TRANSCRIPT
Recombinant DNA Technology
Further Historical Perspective
Geneticists have known for a long time how to isolate DNA from cells.
Geneticists have known for a long time how to chop DNA into small pieces.
What geneticists did not know how to do until the early 1970s was to replicate small fragments of DNA.
1970s Breakthrough
• The discovery of the restriction enzyme
(or restriction endonuclease).
Properties of RE
• Cut double-stranded DNA at specific target sites.
• Allow fragments of DNA that have been cut with the same RE to be rejoined.
AnimationAnimation
• Recombinant DNA_Animation_1
• Recombinant DNA_Animation_2
The joining of two DNA fragments by DNA ligase produces a recombinant DNA molecule
Therefore, eukaryotic DNA could be propagated in prokaryotic cells.
A great breakthrough!!!!!
•
Carriers of foreign DNA are Vectors:
• Most carriers are
• 1. Plasmids
• 2. Bacteriophages (viruses)
• A bacteriophage is a virus that
infects a bacteria.
Introduction to PCR• PCR (polymerase chain reaction)
• PCR is a means to enhance/replicate the amount of DNA collected (in vitro)
* p r c
PCR Creates more DNA for Study
• PCR Animation
DNA Replication Review:
• Add DNA polymerase, all 4 DNA building blocks ???
5’ CTGACGCTGCTGCATGCTAGCT 3’
3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review:
• Primers are required:
5’ CTGACGCTGCTGCATGCTAGCT 3’
CGA 5’
5’ CTG
3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review:
• Primers are required:
5’ CTGACGCTGCTGCATGCTAGCT 3’
. . . t a c g a t CGA 5’
5’ CTG a t g c t g . . . .
3’ GACTACGACGACGTACGATCGA 5’
Introduction to Agarose Gel Electrophoresis
Weigh out ~ a gram of agarose.
• Mix the agarose with 50- 100 ml of buffer.
• Heat to dissolve the agarose.
• Assemble the gel tray and comb.
Pour the gel.
• Pick up the DNA sample with a micro-pipettor.
• Load one DNA sample into each well on the gel.
Connect the gel to a low voltage power supply.
After completion of the run, add a DNA staining material and visualize the DNA
under UV light.
• Analyze the results.
• Gel Electrophoresis – demonstration
• Ensure you’re plugged into the web ;)
• Electrophoresis Demo 1 – flash simulation
• Electrophoresis Demo 2 – java applet
• You need to be hooked to the web for these to work ;)