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Functional Genomics Research Stream
Lecture: April 28, 2009Continued Understanding of PCR Verification
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Quick Issues
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Functional Genomics 2009 T Shirts
$15 EachSee Robbie & Andrew
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Reminder:Bonus Seminar II
• Wednesday, April 29 - 12:00 PM - PAI 4.28
• Course: NSC 109
• Dr. David Brown:Perspective: Industrial Biotechnology Careers
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Current Issues
• Citizenship & Cleaning
• Fall Course Registration
• Course Evaluation
• Email Sent to You
• Please Participate Constructively
• Final Report
• Come by Office for Evaluations
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Questions Regarding Final Reports?
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PCR Clarity
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Process Review
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DNA Representation
5’
convenientrepresentation
chemicalrepresentation
3’ 5’3’
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5’
3’ 5’
3’start with double stranded DNA
5’
3’ 5’
3’denaturation
5’
3’ 5’
3’annealing of primers
5’
3’ 5’
3’
elongation by polymerase (first cycle complete)
3’ 5’
5’ 3’
second cycle complete
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
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How Does PCR Relate to Study of Deletion Strains?
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A Gene is Deleted
GENE
KAN
is replaced with
by homologous recombination
chromosome
chromosome
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PCR Verification of Deletion is Possible
chromosome
chromosome
pA pDpB pC
pKanBpA pD
PCR products reverse primerforward primer
pKanC
reverse primerforward primer
GENE
KAN
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GENEchromosome
pA pDpB pC
PCR products
reverse primer
forward primer
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GENE+
pA pDpB pC
-
... A G A T T T A G C T A T C T A G C C T A G A T T A C A ....
... T C T A A A T C G A T A G A T C G G A T C T A A T G T ....
A A A T
pA5’ 3’
5’3’ pB
pC
pD
T A T C C G G A T A C A
10 nucleotides 12 nucleotides
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+
+
-
-
1015203050
c & d
a & b
ladder
GENEchromosome
pA pDpB pC
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KAN+
-
... A G A T T T A G ? ? ? ? ? ? ? ? ? ? ? A G A T T A C A ....
... T C T A A A T C ? ? ? ? ? ? ? ? ? ? ? T C T A A T G T ....
pA5’ 3’
5’3’ pDpB
(not present)
pC(not present)
pA pDpB pC
A A A T T A T C C G G A T A C A
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+
+
-
-
1015203050
c & d
a & b
ladder
a & d
KANchromosome
pA pD
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PCR Outcomes
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
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ACT1chromosome
FP RP
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Understanding as a Whole Unit
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A Pattern ....
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Next Week:The Research Plan
what, when & how
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Questions?