* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Microarray technique and Functional genomics
Cre-Lox recombination wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Gene therapy wikipedia , lookup
Genomic library wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Human genome wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Gene desert wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Point mutation wikipedia , lookup
Epigenomics wikipedia , lookup
Primary transcript wikipedia , lookup
Pathogenomics wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Transposable element wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Genetic engineering wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Oncogenomics wikipedia , lookup
Public health genomics wikipedia , lookup
Metagenomics wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Non-coding DNA wikipedia , lookup
Ridge (biology) wikipedia , lookup
Genome editing wikipedia , lookup
Genomic imprinting wikipedia , lookup
Genome (book) wikipedia , lookup
Gene expression programming wikipedia , lookup
Genome evolution wikipedia , lookup
Minimal genome wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
History of genetic engineering wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Helitron (biology) wikipedia , lookup
Microevolution wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Designer baby wikipedia , lookup
Microarray and functional genomics Wenjing Tao University of Missouri Microarray: high through-put whole genome approach 48 grids, with 31k probes Each grid contain 650 probes Microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern Microarray terminology • Feature - an array element • Probe - a feature corresponding to a defined sequence (immobilized on a solid surface in an ordered array) • Target - a pool of nucleic acids of unknown sequence Microarray provides the opportunities - Find the genes and assign them functions - Predict protein structures and functions - Reconstruct metabolic, signaling, and other pathways - Reconstruct informational networks - Link genotype to phenotype - Use genotype/phenotype to predict relevant outcome - Cross- species comparisons Kinds of array features Synthetic oligonucleotides: Affymetrix genechip Long oligo array PCR products from: Cloned cDNAs Genomic DNA cDNA & oligonucleotide arrays 100-300 m spot 20-25 mers Schulze and Downward, 2001 Nat Cell Biol 3, 190 cDNA and long oligo array experiment RNA Target1 Target2 RT Labeling with Flouresent dye RT Hybridization Scan ORFs or ESTs •••••••••• •••••••••• •••••••••• •••••••••• Design long oligoes Microtiter plate Microarray slides Affymetrix GeneChip RED represents Test DNA hybridized to the target DNA. GREEN represents Reference DNA hybridized to the target DNA. YELLOW represents a combination of Test and Reference DNA hybridized equally to the target DNA. BLACK represents areas where neither the Reference nor Test DNA hybridized to the target DNA. Fluorescent microarrays are composed of a combined two false color laser scanned images Image file post-processing • Single slide normalization – GenePix Pro 4.1 • Slide-slide and dye-swap comparison – TMEV & MIDAS • Cross-slides quality evaluation - GeneSpring + R script for CV filter • Mixed linear model analysis of Variance to identify significant differentially expressed genes – R or SAS program • Data Analysis in the Post-Genomic Era (gene annotation, ontology and pathway analysis– KOG, COG, KEGG, TAIR, Onto-Tools, GenMapp… • Data validation – qPCR or Northern blot Whole genome approaches to biological questions • Gene expression • Gene variation • Gene function Functional Genomics of Root Growth and Root Signaling under drought NSF-DB1-0211842, PI: Henry Nguyen http://rootgenomics.missouri.edu/prgc/research.html Drought-stress inducible genes and their possible functions in stress tolerance and response. Yamaguchi-Shinozaki et al. JIRCAS Working Report, 2002 Characterize the transcript profiles of apical and basal regions of the root growth zone under water deficit condition using maize long oligonucleitide arrays Dr. Henry Nguyen’s lab, Plant Sciences, University of Missouri Objectives To identify genes contributing to root growth maintenance under water deficit condition To determine genes responsible for progressive inhibition of root elongation under water-deficit condition To compare the differential gene expression in root region of progressive inhibition of root elongation under water stress with the normal growth deceleration in well-watered root region Pair-wise comparison of maize root segments using oligo array 4 3 6 5 4 3 2 2 1 1 WW48 4 3 2 1 WS48 Characterization of the maize long oligo array • Maize oligo array, printed at the University of Arizona, contains 56,311 70-mer oligonucleotide probes, including >30,000 identifiable unique maize genes. 16,915 oligoes do not have any annotation. • 70-mer oligonucleotides in conjunction with Operon Qiagen based on the TIGR Maize Database Slides feature and dye-swap experiment WS/WW=Cy5/Cy3 WS/WW=Cy3/Cy5 Dye Swap Two-color microarray data feature 1. Channel A intensity vs. channel B intensity 4. Z-score histogram 2. Log channel A intensity vs. log channel B intensity 5. Box plot 3. R-I Flip dye consistency checking - processed data count: 27852 (only slides A) - pre-filtering corr. coeff: 0.11360581 - post-filtering data count: 26747 - confidence factor: 0.9647781 - dispersion factor: 0.035401408 Summary of the evaluation of replicates (technique & biological) • ~50,000 of the 56,311 genes have intensity >200 (at least one channel). • Confidence of dye-swap is > 96% • 99.9% confidence limit was estimated by testing the coefficient of variance (CV) for replicates Mixed linear model analysis of two color microarray data- producing lists of differentially expressed genes with low false discovery rates To obtain accurate and precise estimates of gene expression values between treatment and control, analyze gene effects with a simultaneous consideration of all blocking factors, a linear mixed ANOVA model is applied: There are two processes: First, global mixed model was applied: Log2(singal values) = treat + dye + treat*dye + tech_reps_effect + array_effect (within treat*dye and tech_reps_effect) Second, take residual values from the first model and then apply this model for individual gene: Residuals = treat + dye + tech_reps_effects + array(within tech_reps_effects) Gene function categorization of significantly differentially expressed genes CELLULAR PROCESSES AND SIGNALING 10% INFORMATION STORAGE AND PROCESSING - 4% METABOLISM - 11% POORLY CHARACTERIZED - 6% NOT ASSIGNED – 69% KOG analysis Information storage and processing Replication, recombination and repair 14% RNA processing and modification 16% Chromatin structure and dynamics 13% Transcription 34% Translation, ribosomal structure and biogenesis 23% Metabolites Secondary metabolites biosynthesis, transport and catabolism 14% Energy production and conversion 15% Amino acid transport and metabolism 14% Inorganic ion transport and metabolism 16% Lipid transport and metabolism 14% Coenzyme transport and metabolism 4% Nucleotide transport and metabolism 5% Carbohydrate transport and metabolism 18% CELLULAR PROCESSES AND SIGNALING Cytoskeleton 6% Nuclear structure 1% Extracellular structures 1% Defense mechanisms 8% Cell cycle control, cell division, chromosome partitioning 3% Cell wall/membrane/envelope biogenesis 9% Cell motility 0% Intracellular trafficking, secretion, and vesicular transport 11% Posttranslational modification, protein turnover, chaperones 30% Signal transduction mechanisms 31% Summary • Microarray is a high through-put tool to identify novel genes • We have identified 19 hundred drought response and root growth maintenance related genes • Combining functional analysis we would find drought stress tolerance related pathways and genes • This knowledge will lead to novel approaches for improving drought tolerance in maize.