Methods for Gene Activity Analysis

1. Methods for Gene Activity Analysis By Auni Hovanesian Krista Templeton

2. What Methods Can We Use to Study Gene Expression? • Three Basic Approaches: • RT-PCR • GeneChip Microarray • Upstream Regulatory Region Analysis

3. What Is The Basis of RT-PCR Activity Analysis? mRNA! • mRNA is only present in cells for genes that have been transcribed and will usually be actively expressed once translated into proteins • RT-PCR links gene expression to mRNA presence in different compartments of plant • But in order to trace mRNA back to an original gene, must amplify sample, since often only small amounts present

4. Why RT-PCR Rather Than Regular PCR? • mRNA will not work in our PCR reaction, so need to convert it to cDNA • reagents being used in PCR are DNA-specific (i.e. DNA polymerase) • DNA is more stable than RNA • More practical for long-term storage purposes • RNA’s instability would give lower yield in PCR

5. AAAAAAA So How Does RT-PCR Work? • Isolate mRNA from area-specific plant tissue/organ samples • Convert all isolated mRNA strands to cDNA using Reverse Transcriptase mRNA Reverse Transcriptase cDNA

6. 3) Use first cDNA template as now compatible basis of PCR: • Only need to synthesize a single strand of cDNA template to start PCR • One cDNA representing every mRNA in each sample--like gene expression library • PCR is like selection of a particular gene from the library (if it is there!) • Gene-specific primers designed in exons (due mRNA splicing) • If PCR product formed with gene-specific primers for RT-PCR, will have amplified the cDNA correlate of original gene • Indicates presence of mRNA correlating to gene of interest • Helps localize gene activity to wherever sample came from

7. Sample RT-PCR Results 100 bp Silique RT+ Positive Control Negative Control Silique RT- Leaf RT+ Leaf RT- Tubulin Bands 475 bp Hypothetical cDNA Band 200 bp

8. So How Do We Verify and Further Specify RT-PCR Results? GeneChip Microarray Analysis!

9. What is a GeneChip Microarray? • Using cDNA created from the mRNA isolated from various organs, we can analyze the mRNA accumulation levels for all genes • Done by creating the complementary strands of all the known gene sequences and assembling them on a chip • Different chips are used for various stages of development • The cDNA sequences are tagged with flourescent labels that glow a certain color when in contact with the complementary strand; colors read by a computer

10. What is the Resulting Image? Red means active Green means not as active No color means no activity

11. What Does the Online GeneChip Report Look Like?

12. So, How Do RT-PCR and GeneChips Complement Each Other? • RT-PCR may be slightly more accurate, since working with smaller fragments (only portions of cDNA) • But GeneChips provide more specificity more efficiently • The two combined provide convenient checkpoints for each other’s results • both working with same principle of mRNA level analysis to determine gene expression

13. What Is Another Method of Gene Expression Analysis?

14. Upstream Region Analysis

15. Overview • The upstream regulatory region of a gene contains its “on” switch. • Once the upstream region is fused to GFP (green fluorescent protein) or GUS (betaglucuronidase) it can be transformed into the Arabidopsis plant • Once transformation has occurred gene expression will indicate where the gene of interest is transcribed.

16. Strategy of Promoter Activity Analysis • Arabidopsis Genomic DNA • PCR amplification of upstream region • With Gene-specific Primers • And High Fidelity DNA Polymerase PCR Product pENTR/D-TOPO vector Ligation: Population of Recombinant Plasmid (vector+PCR product) and NON-recombinant plasmid (vectory only) Transformation of competent E.coli cells Screening for E.coli cells harboring recombinant plasmid Confirmed Recombinant plasmid DNA: Verifying the authenticity of recombinant plasmid DNA by Restriction Enzyme Digestion Recombinant Plasmid DNA + Beta-Gluronidase (GUS) gene carrying T-DNA Vector DNA sequences: verification of the cloned Promoter Region by Sequencing Analysis. Sequence Analysis and confirmed identity of the cloned upstream region

17. What Are the Steps Required to Isolate the Promoter Region? • Conduct PCR on the Arabidopsis DNA in order to amplify the upstream region with iProof polymerase

18. Why Use a Proofreading Polymerase? • iProof polymerase corrects nucleotide errors during amplification • One mutation could affect the transcription of a gene

19. What To Do After the Promoter Region Has Been Ampified • The section needs to be enzymatically inserted into a plasmid vector where it can be placed into an E.coli cell. DNA pENTR-TOPO vector Recombinant Plasmid Mix E.coli cells with plasmids in presence of CaCl. Culture on nutrient agar plates containing ampicillin.

20. What Does Topoisomerase 1 Do? • The pENTR/d-TOPO vector contains Topoisomerase 1 • Relieves supercoils in circular DNA plasmids by nicking one of the strands of the DNA double helix • Linearized the pENTR vector, allowing insertion of the PCR fragment • Re-ligates vector

21. How Do GUS and GFP Work and What Are Their Differences? • A mature Arabidopsis embryo expressing Green Fluorescent Protein GUS is the more sensitive of the two Common Reporter Genes • GUS and GFP connect to the promoter region. When the genes are turned on through the promoter region GUS and GFP are turned on also

22. Acknowledgements • Jordan, Jennifer, and Brian from Spring 2006 for some useful slide ideas and diagrams • Kelli for her presentation on Upstream Region Analysis • Brandon for all his tech support! • Anhthu, Bekah, and Daisy for their help and many explanations