Analysis of Gene Expression of Arabidopsis using RT-PCR and

DNA Cloning

Presented by Neha Jain

ABE Workshop 2006

June 30, 2006

AIM: To isolate and sequence the cDNA of PDI2 and GFP

- Extraction of total RNA from Arabidopsis - RNA template cDNA Amplify using PCR

- Run gel using RT-PCR products

- Purification and quantification of the cDNA

- Ligation of PCR product into a TA vector

- Amplification of vector DNA in bacteria

Introduction

RT

Methods

Isolation of total RNA (rRNA, tRNA, mRNA)

- Pulverize the leaves into a fine powder

- Lysis of the cell under denaturing conditions

- Centrifuged to separate the RNA from other cellular materials

- The lysate is applied to a column that binds RNA

- The RNA is eluted with water

Reverse Transcription

• Reverse Transcriptase (RT) – a DNA polymerase enzyme that uses an RNA template to synthesize a complementary molecule of double stranded DNA

• We used Moloney murine leukemia virus (M-MLV) for the synthesis of the first cDNA strand

mRNA Oligo dT primer

cDNA

Methods

Reverse Transcription

- The following samples were used:

• The concentration of RNA added to tubes 1-4 was 2μg

Group 1 2 3 4 5

1,4 WT PDI2 A-1

PDI2 A-2

No RT Control- No RNA

2,3 WT pBIN35S-GFP

pGFP 2SC-1

No RT Control- No RNA

Methods • The following reagents were added to the RNA samples (which

contain template mRNA)

1. RT Buffer

2. RNase-free DNase

3. dNTPs

4. Oligo dT Primers

5. RNase-free water

37ºC – 30 min

Activate DNase

70ºC – 5 min

Inactivate DNase

RT Reaction and PCR RT:

PCR:

42ºC - 1 hour

Add RT

95ºC - 5 min

Template DNA

PCR buffer

MgCl2

dNTPs

Gene specific primers

DNA Polymerase

PCRProduct

Run on gel

95ºC 5 min

denature

95ºC – 30 sec 56ºC – 30 sec 72ºC – 1 min

72ºC 5 min

Anneal and

elongate

35 CYCLES

Anneal and

elongate

Results of RT-PCR

Group 1 Group 4

100bp + WT 2A1 2A2 –RT H2O P+ 100bp + WT 2A1 2A2 –RT H2O P+

Summary of RT-PCR Results

Groups 1 and 4

Lane Sample Band?

G1 G4

Explanation or Expected Result

2 Henry’s RT-PCR Positive Control

Henry’s RT-PCR

3 WT X WT has the PDI2 gene

4 PDI2 A-1 X X Homozygous – PDI2 gene

knocked out

5 PDI2 A-2 X X Heterozygous – should see band

6 (-) No RT X X No RT

7 (-) Control H20 X X No RNA template

8 Henry’s Plasmid Control PDI2 inserted into plasmid

Results of RT-PCR

100bp + WT 2SC GFP5er –RT H2O P+

Group 2

100bp + WT 2SC GFP5er –RT H2O P+

Group 3

Summary of RT-PCR Results

Groups 2 and 3

Lane Sample Band?

G2 G3

Explanation or Expected Result

2 Henry’s Positive Control Henry’s RT-PCR

3 WT X X GFP absent in WT

4 GFP2SC X X Band expected - transgenic plant

5 GFP5er (35S) GFP inserted into plant

6 (-) No RT X X No RT

7 (-) Control H20 X X No RNA template

8 Henry’s Plasmid Control GFP inserted into plasmid

Methods

Extraction and Purification of DNA from the Agarose Gel - Cut the selected DNA fragment from the agarose gel

- Melted the gel

- The sample was applied to a column where the DNA binds to the membrane of the column

- The DNA is eluted with buffer

- The DNA can then be cloned using plasmid vectors

RT-PCR Products Selected for TOPO Cloning

MW Hen Pl GFP H+ pBIN GFP WT Pl+ PDI2 PDI2 35S 35S pla

Gr 1 Gr2 Gr3 Gr4

Gel purified RT-PCR products for use as inserts in

cloning

Molecular Cloning

What is Molecular Cloning?

Refers to making multiple copies of a gene or fragment of DNA by insertion of the DNA fragment into a vector which can then replicate in a host cell

Requirements for CloningForeign DNA

- gel purified RT-PCR products

Vector DNA

- pCR4-TOPO plasmid vector

Host Organism

- E. coli

Overview of DNA Cloning Using a Plasmid Vector

TOPO TA CloningUses DNA Topoisomerase I

- it cleaves and re-joins DNA during replication

- functions as a restriction enzyme and a ligase

- covalently bonded to 3' deoxythymidine (T) that overhangs a linearized plasmid

- complements the 3' deoxyadenosine (A) that overhangs the PCR product

Methods - TOPO TA Cloning

Ligation of the product into a TA Plasmid Vector

- add the gel purified PCR product into 2 tubes and H20 in the third as a control

- add the pCR4 TOPO Vector to each tube

- add salt solution to each tube which helps to ligate the DNA

H20 PCR Product 1 PCR Product 2

pCR4 TOPO Vector

Plasmid Vector

Transformation of E. coli

• Transformation – the uptake of foreign DNA by bacterial cells

• The TOPO vector with the insert is added to “competent” E. coli

• The E. coli are made “competent” by treating them with ice-cold CaCl2 followed by heat-shock at 42ºC

• S.O.C medium is added to the bacteria and then incubated at 37ºC for 1 hour during which transformation takes place

Amplification of the Recombinant Plasmid in Bacteria

• Host bacteria are spread on a selective nutrient medium and incubated at 37ºC overnight

• The plates contain ampicillin which allow the bacteria containing the recombinant plasmid to proliferate forming colonies

• E. coli divide every 22 minutes forming a billion cells in less than 11 hours

• The recombinant plasmids also replicate as the bacteria divide

Selection of Cells Containing Insert

Insertional Mutagenesis

- The LacZ gene in the plasmid codes for β-galactosidase

- Xgal is a substrate for the enzyme which turns from colorless to blue

White Colonies

- β-galactosidase not produced due to insert

Blue Colonies

- β-galactosidase produced as there is no insert

Selection of Cells Containing Insert

pCR4-TOPO Vector

- the lethal E. coli ccdB gene in the pCR4-TOPO plasmid is responsible for killing the cells without an insert

Ligation Efficiency of RT-PCR Products into the pCR4 Vector

Number of Colonies

Volume

plated

Group1 Group 2 Group 3 Group 4

Water

Negative

10 uL 52 60 96 7750 uL 220 171 258 225

Ligation 1 10 uL 83(Henry positive)

136(Henry positive)

65(Henry positive)

45(wild type)

50 uL 672(Henry positive)

600(Henry positive)

440(Henry positive)

145(wild type)

Ligation 2 10 uL 98(pdi2)

107(35Ser)

33(35Ser)

17 (white)

6 (blue)

50 uL 412(pdi2)

515(35Ser)

207(35Ser)

513(pdi2)

Ligation Efficiency of RT-PCR Products into the pCR4 Vector

Each group picked 10 white colonies from both ligation 1 and 2 and placed them into 10 different tubes containing LB and Ampicillin which were cultured overnight

Results

Group Number of tubes with

growth

Total number of tubes

1 9 10

2 1 10

3 1 10

4 4 10

Total 15 40

Any Questions?

References

• Becker, W. M., Kleinsmith L.J., Hardin, J. (2006). The world of the cell, San Francisco: Pearson Education

• http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring99/bill/invitrogen.html

• http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/clones.html

• http://en.wikipedia.org/wiki/Cloning_vector

• http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/rt.html

• http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf