Recombinant DNA Technology. Further Historical Perspective Geneticists have known for a long time...
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Transcript of Recombinant DNA Technology. Further Historical Perspective Geneticists have known for a long time...
![Page 1: Recombinant DNA Technology. Further Historical Perspective Geneticists have known for a long time how to isolate DNA from cells. Geneticists have known.](https://reader031.fdocuments.us/reader031/viewer/2022033107/56649e0c5503460f94af5b6f/html5/thumbnails/1.jpg)
Recombinant DNA Technology
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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.
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1970s Breakthrough
• The discovery of the restriction enzyme
(or restriction endonuclease).
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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.
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AnimationAnimation
• Recombinant DNA_Animation_1
• Recombinant DNA_Animation_2
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The joining of two DNA fragments by DNA ligase produces a recombinant DNA molecule
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Therefore, eukaryotic DNA could be propagated in prokaryotic cells.
A great breakthrough!!!!!
•
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Carriers of foreign DNA are Vectors:
• Most carriers are
• 1. Plasmids
• 2. Bacteriophages (viruses)
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• A bacteriophage is a virus that
infects a bacteria.
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Introduction to PCR• PCR (polymerase chain reaction)
• PCR is a means to enhance/replicate the amount of DNA collected (in vitro)
* p r c
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PCR Creates more DNA for Study
• PCR Animation
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DNA Replication Review:
• Add DNA polymerase, all 4 DNA building blocks ???
5’ CTGACGCTGCTGCATGCTAGCT 3’
3’ GACTACGACGACGTACGATCGA 5’
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DNA Replication Review:
• Primers are required:
5’ CTGACGCTGCTGCATGCTAGCT 3’
CGA 5’
5’ CTG
3’ GACTACGACGACGTACGATCGA 5’
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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’
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Introduction to Agarose Gel Electrophoresis
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Weigh out ~ a gram of agarose.
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• Mix the agarose with 50- 100 ml of buffer.
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• Heat to dissolve the agarose.
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• Assemble the gel tray and comb.
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Pour the gel.
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• Pick up the DNA sample with a micro-pipettor.
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• Load one DNA sample into each well on the gel.
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Connect the gel to a low voltage power supply.
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After completion of the run, add a DNA staining material and visualize the DNA
under UV light.
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• Analyze the results.
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• Gel Electrophoresis – demonstration
• Ensure you’re plugged into the web ;)
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• Electrophoresis Demo 1 – flash simulation
• Electrophoresis Demo 2 – java applet
• You need to be hooked to the web for these to work ;)