Methods for Gene Activity Analysis By Auni Hovanesian Krista Templeton.

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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