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?