functional genomics research...

Functional Genomics Research Stream

Lecture: April 28, 2009Continued Understanding of PCR Verification

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• Wednesday, April 29 - 12:00 PM - PAI 4.28

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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?