methods for gene activity analysis by auni hovanesian krista templeton
TRANSCRIPT
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Methods for Gene Activity Analysis
By Auni Hovanesian Krista Templeton
What Methods Can We Use to Study Gene Expression?
• Three Basic Approaches:1) RT-PCR2) GeneChip Microarray3) Upstream Regulatory
Region Analysis
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What Is The Basis of RT-PCR Activity Analysis?
• 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
mRNA!
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
So How Does RT-PCR Work?
1) Isolate mRNA from area-specific plant tissue/organ samples
2) Convert all isolated mRNA strands to cDNA using Reverse Transcriptase
AAAA
AAA
Reverse Transcriptase
mRNA
cDNA
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
Sample RT-PCR Results
Leaf RT+ Silique RT+
Silique RT-
Negative Control
Leaf RT- Positive Control
100 bp
Tubulin Bands 475 bp
Hypothetical cDNA Band 200 bp
So How Do We Verify and Further Specify RT-PCR
Results?
GeneChip Microarray Analysis!
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
What is the Resulting Image?
Red means active
Green means not as active
No color means no activity
What Does the Online GeneChip Report Look Like?
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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
What Is Another Method of Gene Expression
Analysis?
Upstream Region AnalysisUpstream Region Analysis
OverviewOverview
• The upstream regulatory region of a The upstream regulatory region of a gene contains its “on” switch.gene contains its “on” switch.
• Once the upstream region is fused to Once the upstream region is fused to GFP (green fluorescent protein) or GFP (green fluorescent protein) or GUS (betaglucuronidase) it can be GUS (betaglucuronidase) it can be transformed into the Arabidopsis transformed into the Arabidopsis plantplant
• Once transformation has occurred Once transformation has occurred gene expression will indicate where gene expression will indicate where the gene of interest is transcribed.the gene of interest is transcribed.
Strategy of Promoter Activity Strategy of Promoter Activity AnalysisAnalysis
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
DNA sequences: verification of the cloned Promoter Region by Sequencing Analysis. Sequence Analysis and confirmed identity of the cloned upstream region
Recombinant Plasmid DNA + Beta-Gluronidase (GUS) gene carrying T-DNA Vector
What Are the Steps Required What Are the Steps Required to Isolate the Promoter to Isolate the Promoter
Region?Region?• Conduct PCR on the Arabidopsis DNA Conduct PCR on the Arabidopsis DNA
in order to amplify the upstream in order to amplify the upstream region with iProof polymeraseregion with iProof polymerase
Why Use a Proofreading Why Use a Proofreading Polymerase?Polymerase?
• iProof polymerase corrects iProof polymerase corrects nucleotide errors during amplificationnucleotide errors during amplification
• One mutation could affect the One mutation could affect the transcription of a genetranscription of a gene
What To Do After the Promoter What To Do After the Promoter Region Has Been AmpifiedRegion Has Been Ampified
• The section needs to be The section needs to be enzymatically inserted into a plasmid enzymatically inserted into a plasmid vector where it can be placed into an vector where it can be placed into an E.coli cell.E.coli cell.
pENTR-TOPO vector
DNA
Recombinant Plasmid
Mix E.coli cells with plasmids in presence of CaCl. Culture on nutrient agar plates containing ampicillin.
What Does Topoisomerase 1 What Does Topoisomerase 1 Do?Do?
• The pENTR/d-TOPO vector The pENTR/d-TOPO vector contains Topoisomerase 1contains Topoisomerase 1– Relieves supercoils in Relieves supercoils in
circular DNA plasmids circular DNA plasmids by nicking one of the by nicking one of the strands of the DNA strands of the DNA double helixdouble helix
– Linearized the pENTR Linearized the pENTR vector, allowing vector, allowing insertion of the PCR insertion of the PCR fragmentfragment
– Re-ligates vectorRe-ligates vector
How Do GUS and GFP Work How Do GUS and GFP Work and What Are Their and What Are Their
Differences?Differences?•A mature A mature ArabidopsisArabidopsis embryo expressing Green embryo expressing Green Fluorescent ProteinFluorescent Protein
GUS is the more sensitive GUS is the more sensitive of the two Common of the two Common Reporter GenesReporter Genes
•GUS and GFP connect to the GUS and GFP connect to the promoter region. When the genes promoter region. When the genes are turned on through the are turned on through the promoter region GUS and GFP are promoter region GUS and GFP are turned on alsoturned on also
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