Analysis of Gene Expression of Arabidopsis using RT-PCR and DNA Cloning

1. Analysis of Gene Expression of Arabidopsis using RT-PCR and DNA Cloning Presented by Neha Jain ABE Workshop 2006 June 30, 2006

2. Introduction 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 RT

3. 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

4. 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

5. Methods Reverse Transcription - The following samples were used: • The concentration of RNA added to tubes 1-4 was 2μg

6. 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 70ºC – 5 min Activate DNase Inactivate DNase

7. RT Reaction and PCR Add RT RT: 95ºC - 5 min 42ºC - 1 hour Anneal and elongate PCR: Template DNA PCR buffer MgCl2 dNTPs Gene specific primers DNA Polymerase 95ºC – 30 sec 56ºC – 30 sec 72ºC – 1 min 95ºC 5 min 72ºC 5 min PCR Product denature Anneal and elongate Run on gel 35 CYCLES

8. Results of RT-PCR 100bp + WT 2A1 2A2 –RT H2O P+ 100bp + WT 2A1 2A2 –RT H2O P+ Group 1 Group 4

9. Summary of RT-PCR Results Groups 1 and 4

10. Results of RT-PCR 100bp + WT 2SC GFP5er –RT H2O P+ 100bp + WT 2SC GFP5er –RT H2O P+ Group 2 Group 3

11. Summary of RT-PCR Results Groups 2 and 3

12. 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

13. RT-PCR Products Selected for TOPO Cloning Gr 1 Gr2 Gr3 Gr4 MW Hen Pl GFP H+ pBIN GFP WT Pl+ PDI2 PDI2 35S 35S pla Gel purified RT-PCR products for use as inserts in cloning

14. 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 Cloning Foreign DNA - gel purified RT-PCR products Vector DNA - pCR4-TOPO plasmid vector Host Organism - E. coli

15. Overview of DNA Cloning Using a Plasmid Vector

16. TOPO TA Cloning Uses 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

17. 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 pCR4 TOPO Vector H20 PCR Product 1 PCR Product 2

18. Plasmid Vector

19. 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

20. 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

21. 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

22. 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

23. Ligation Efficiency of RT-PCR Products into the pCR4 Vector

24. 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

25. Any Questions?

26. 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