functional genomics research...
TRANSCRIPT
Functional Genomics Research Stream
Lecture: April 28, 2009Continued Understanding of PCR Verification
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PCR Clarity
Process Review
DNA Representation
5’
convenientrepresentation
chemicalrepresentation
3’ 5’3’
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’
How Does PCR Relate to Study of Deletion Strains?
A Gene is Deleted
GENE
KAN
is replaced with
by homologous recombination
chromosome
chromosome
PCR Verification of Deletion is Possible
chromosome
chromosome
pA pDpB pC
pKanBpA pD
PCR products reverse primerforward primer
pKanC
reverse primerforward primer
GENE
KAN
GENEchromosome
pA pDpB pC
PCR products
reverse primer
forward primer
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
+
+
-
-
1015203050
c & d
a & b
ladder
GENEchromosome
pA pDpB pC
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
+
+
-
-
1015203050
c & d
a & b
ladder
a & d
KANchromosome
pA pD
PCR Outcomes
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
+
+
-
-
a & b
ladder
+ ctrl
KANchromosome
pA pD
GENEchromosome
pA pDpB pC
pKanB pKanC
- ctrl
a & kanB
ACT1chromosome
FP RP
Understanding as a Whole Unit
A Pattern ....
Next Week:The Research Plan
what, when & how
Questions?