Functional Genomics Research...
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Functional Genomics Research Stream Research Meeting: February 28, 2012 PCR Optimization, PCR Mediated Gene Deletion, Transformation of Yeast Screening for Gene Deletions by PCR
Transcript of Functional Genomics Research...
Functional Genomics Research StreamResearch Meeting: February 28, 2012
PCR Optimization, PCR Mediated Gene Deletion, Transformation of YeastScreening for Gene Deletions by PCR
Lab Issues & Progress• RPR II & III Due:
Friday, March 9 @ 12:00PMHand in during Mar. 6th class or Mailbox ....
• Lab Open & Mentors Present10am to 8pm (M, W, Th, F), 3pm to 7 pm (Sat)
• Tuesdays for Quick Maneuvers ....
• Research Resource Calendars(Bench & PCR)
Lab Issues• Incubator: Don’t wrap plates, throw out old
plates and cultures
• Waste: phenol and ethidium bromide
• Hood: Use pH meter at bench instead • Be sure to screw cap on HCl, NaOH• Clean up after yourself
• Rinse flask out roughly after pouring gel
• Remember DNA/RNA precipitations must be done in 4 ºC centrifuge
PolymeraseChain Reaction
PrimersT A
CG
TT T A T ATT T A T ATT T A Tm = 28 ºC
18 mersG CG G CG G CG G CG G CG G Tm = 76 ºC
T A
CG
TT T A T ATT T A T ATT T A
Tm = 36 ºC24 mersG CG G CG G CG G CG G CG G
Tm = 84 ºC
T ATT T A
CG G CG G
5’
5’
5’
5’
3’
3’
3’
3’
Mis-priming of PrimersA GT CGT C GTC G TC A T A C C T T T CCAAACCC 5’
5’3’
3’T A CGTT T
A GT CGT C GTC G TC A T A C C T T T CCAAACCC
5’
3’
Correct
5’T A CG
TT
T
T
T
3’
Mispriming
A GT CGT C GTC G TC A T A C C T T T CCAAACCC3’ 5’T
5’ 3’A C
TGT
T T
Creation of Primer Dimers
5’ 3’T A CGTT T
5’ 3’T A CGTT T
5’3’ TAC G T TT
5’ 3’T A CGTT T
5’3’ TAC G T TT
5’ 3’T A CGTT T
5’3’ TAC G T TT
5’ 3’T A CGTT T A A AAAA
PCR
What if your PCR looks like this?
How can we favor amplification of product with thermocycler conditions?
58°C0:30
94°C5:00
94°C0:30
72°C0:30
25 cycles
72°C7:00 4°C
∞
We want to make a 1.5 kb product. The primers have
Tm of 58°C and 51°C
250
5007501000
1500
bp
E NTC
PCR
What if your PCR looks like this?
How can we favor amplification of product with thermocycler conditions?
58°C0:30
94°C5:00
94°C0:30
72°C0:30
25 cycles
72°C7:00 4°C
∞
We want to make a 1.5 kb product. The primers have
Tm of 58°C and 51°C
250
5007501000
1500
bp
E NTC
PCR
PCR components:•genomic DNA•primers•buffer•dNTPs•MgCl2•Taq DNA polymerase
What if your PCR looks like this?
How can we favor amplification of product with reaction conditions?
250
5007501000
1500
bp
E NTC
PCR
PCR components:•genomic DNA•primers•buffer•dNTPs•MgCl2•Taq DNA polymerase
What if your PCR looks like this?
How can we favor amplification of product with reaction conditions?
250
5007501000
1500
bp
E NTC
PCR
What if your PCR looks like this?
How can we favor amplification of the specific product with thermocycler conditions?
51°C0:30
94°C5:00
94°C0:30
72°C1:00
35 cycles
72°C10:00 4°C
∞
We want to make a 1.5 kb product. The primers have
Tm of 58°C and 51°C
250
500750
1000
1500
bp
E NTC
PCR
What if your PCR looks like this?
How can we favor amplification of the specific product with thermocycler conditions?
51°C0:30
94°C5:00
94°C0:30
72°C1:00
35 cycles
72°C10:00 4°C
∞
We want to make a 1.5 kb product. The primers have
Tm of 58°C and 51°C
250
500750
1000
1500
bp
E NTC
PCR
PCR components:•genomic DNA•primers•buffer•dNTPs•MgCl2•Taq DNA polymerase
What if your PCR looks like this?
How can we favor amplification of the specific product with reaction conditions?
250
500750
1000
1500
bp
E NTC
PCR
PCR components:•genomic DNA•primers•buffer•dNTPs•MgCl2•Taq DNA polymerase
What if your PCR looks like this?
How can we favor amplification of the specific product with reaction conditions?
250
500750
1000
1500
bp
E NTC
PCR
PCR components:•genomic DNA•primers•buffer•MgCl2•Taq DNA polymerase
What if your PCR looks like this?
How can we favor amplification of product with reaction conditions?
250
500750
1000
1500
bp
E NTC
A Gene is Deleted
Gene X
kanMX4
is replaced with
Gene X
by transformation & homologous recombination
chromosome
chromosome
How was the gene deleted?
E. coli cells in LB Mediachromosomal DNA
plasmid DNApFA6-KanMX6
E. coli cell
pFA6-KanMX6
AmpR polypeptide coding sequence
KanR polypeptidecoding sequence
E. coli cells in LB+kanamycin Mediachromosomal DNA
plasmid DNApFA6-KanMX6
E. coli cell
Critical Question
• We wish to delete Gene X from the yeast genome.
• This will happen in a process where a very small fraction of cells will actually have the gene deleted.
• How can we design the deletion process to use the process of selection to select for yeast cells that have had Gene X deleted?
Answer
• We will delete Gene X.
• We will replace it with kanMX4 by virtue of the cell’s homologous recombination.
• We will select with the eukaryotic version of kanamycin (geneticin, G418).
• We will analyze the genomic DNA of cells that survive in order to prove Gene X is indeed gone and kanMX4 is present (at
A Gene is Deleted
Gene X
kanMX4
is replaced with
Gene X
by transformation & homologous recombination
chromosome
chromosome
The process.
Gene Deletion1. Use PCR to build DNA fragment you wish to incorporate into the
genome. This fragment must contain a selectable marker.
2. Transform fragment into live cells.
3. Allow cells to incubate with fragment - homologous recombination occurs.
4. Plate cells on selective mediate (YPD+G418).
5. Allow cells to grow for 48 to 72 hours.
6. Select and label colonies, grow overnight culture for each colony selected.
7. Prepare genomic DNA for each overnight, quantitate.
8. Run PCR reactions to interrogate genomic DNA for existence of original gene, incorporation of kanMX4 fragment.
9. Analyze PCR reactions by gel electrophoresis.
2. Use PCR to build DNA fragment you wish to incorporate into the genome. This fragment must contain a selectable marker.
KanMX4
KanMX4
produces
Round 1 PCRUPTAG primer
DOWNTAG primer
PCR purification•Add reagent to PCR after gel verification•Add PCR/reagent mix to binding column•Spin column (DNA remains bound to matrix)•Wash column (what are we removing?)•Elute DNA•Transform yeast cells
3. Transform fragment into live cells.
yeast cell
4. Allow cells to incubate with fragment - homologous recombination occurs.
Gene XCHR III
KanMX4
yeast cell
CHR III KanMX4
AAAAAAAAAAAAAAA
AAAAAAAAAAAAAAA
AAAAAAAAAAAAAAA
AAAAAAAAAAAAAAA
AAAAAAAAAAAAAAA
yeast cell
5. Plate cells on selective mediate (YPD+G418).
AAAAAAAAAAAAAAAAAA
AAAAAAAAA
AAAAAAAAAAAAAAAAAA
yeast cell
YPD + G418 plate
cells that surviveG418 treatmentproduce a colony
6. Allow cells to grow for 48 to 72 hours.
Selective Media: YPD+G418
Transformed Cells on YPD Transformed Cells on YPD+G418
7. Select and label colonies, grow overnight culture for each colony selected.
8. Prepare genomic DNA for each overnight, quantitate.
YPD+G418
OvernightCultures
Genomic DNAPreparations
9. Run PCR reactions to interrogate genomic DNA for existence of original gene, incorporation of kanMX4 fragment.
+R+F
+R+F
kanMX4
Gene Xchromosome
chromosome
pA pDpB pC
pKanBpA pD
PCR product
pKanC
gene Y
gene Y
~500 bp ~500 bp
~500 bp ~500 bp
~500 bp
~500 bp
10. Analyze PCR reactions by gel electrophoresis.
kanMX4
chromosome
chromosome
pA pDpB pC
pKanBpA pDpKanC
~500 bp ~500 bp
~500 bp ~500 bp
Gene X
1 7654321. ladder2. negative control3. positive control4. pD & pKanC5. pA & pKanB6. pC & pD7. pA & pB
2200
1000
500
250
What can we conclude?
kanMX4
chromosome
chromosome
pA pDpB pC
pKanBpA pDpKanC
~500 bp ~500 bp
~500 bp ~500 bp
Gene X
1 7654321. ladder2. negative control3. positive control4. pD & pKanC5. pA & pKanB6. pC & pD7. pA & pB
2200
1000
500
250
What can we conclude?
