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Functional Genomics Lecture 3

Slides borrowed from: Debbie Laudencia-Chingcuanco, USDA-ARS Cheryl Seaton, USDA-ARS Victoria Carrollo, USDA-ARS Zjelka McBride, UC Davis

Functional Genomics

· "Expression" = Transcription (mRNA) AND/OR Translation (protein) · "True" Expression results in a functional gene product

Functional Genomics

· mRNA (transcription) function

­ Northern blotting ­ Complementary DNA (cDNA) libraries

· Expressed sequence tag (EST) libraries · cDNA Microarrays (can be genomic / oligo)

Functional Genomics

· Protein function

· · · · SDS PAGE Western Antibody-based arrays Good ol' structure/function determination

­ X-ray crystallization, NMR, etc.

RNA

PROTEIN

GENOME TRANSCRIPTOME

TRANSCRIPTION

TRANSLATION

PROTEOME

What is cDNA ?

Exon 1 Exon 2 Exon 3

Genomic DNA

transcription

hnRNA

splicing

mRNA

Reverse transcription

protein

cDNA

What is a microarray?

· Orderly grid or matrix of genes (probes) · Genes at each spot identified · Interrogated with labeled RNA · Expression patterns in control vs. treated can be discerned using 2 dyes

RED - OVEREXPRESSED GREEN - UNDEREXPRESSED YELLOW - NO CHANGE

Gene expression analysis using a DNA microarrays

mRNA TEP 1 cDNA

DNA microarray

P.O.Brown & D.Botstein, Nature Genetics Supplement, Vol 21, Jan 1999, pp 33-37

Wheat development

Wheat Grain

Starch and Sucrose Metabolism

Microarray schematic

PRINTING

· Omnigrid microarrayer · 100 slides · 74 384 well plates · 24 pins

PRINTING CONSIDERATIONS (METHOD DEVELOPMENT) · · · · PINS TYPE OF SLIDE (COATING) DNA SOLVENT (SSC OR DMSO) PATTERN DESIRED

Pins

Type of Pin

Microspotting pin allows multiple stamps (>100stamps/dip) Dips into solution ­ pick up 0.2-1.0 ul (depends on the size of the pin) Delivers ­ approx. 1nl of solution We will use a DNA concentration of 0.5ug/ul giving a spot of 0.5ng.

Substrate Coupling Chemistry

1. Non-covalent

2. Covalent

SLID E C O A TIN G

Spot Morphology

Aldehyde, 2XSSC

Spot Morphology vs. Slide Coating (2X SSC)

polylysine

silane

aldehyde

LIST OF CONTROLS

Housekeeping Genes Actin Alpha-Tubulin 1 Alpha-Tubulin 2 Alpha-Tubulin 3 Cytochrome P-450 monooxygenase Elongation Factor 1-alpha EF 1-beta EF1-gamma EF 2 Enolase Glutathione S-transferase Heat Shock Protein (hsp70) Mitochondrial F-1-ATPase subunit 2 Ribosomal Protein L3 RP L7 RP S7 RP S4 S-adenolsylmethionine decarboxylase S-adenolsylmethionine synthetase Ubiquitin Polyubiquitin Lipid Transfer Protein Starch Synthase Starch Synthase I Starch Synthase II Starch Synthase III Starch Branching Enzyme I Starch Branching Enzyme II ADP glucose pyrophosphorylase small subunit ADP glucose pyrophosphorylase large subunit UDP glucose pyrophosphorylase Alpha 1,4-glucan phosphorylase Vacuolar invertase Cell-wall invertase Sucrose synthase I Sucrose synthase II Sucrose phosphate synthetase Sucrose phophatase Sucrose transporter Sucrose fructan 6 -fructosyltransferase Sucrose UDP glucosyltransferase Histone 1 Histone 2 Histone 3 Histone 4 Rubisco small subunit Beta-amylase

Plasma membrane Ca2+-ATPase

H+ transporting ATPase Aldolase Cytoplasmic aldolase Tissue Specific Markers Alpha/Beta gliadin Gamma gliadin Omega gliadin HMW glutenin LMW glutenin Ferredoxin PS II protein PS I protein Oxygen-evolving complex Chlorophyll a/b binding complex

MORE CONTROLS

Negative controls Salmon sperm DNA Endochitinase Exochitinase Trichothecene acetylase GFP fused to DON mimotope AFGC Control Set Transgenes BT: B. Thuringiensis cry1 AC BAR: Phosphinothricin acetyltransferase GFP: Green Fluorescent Protein Globin GUS: Beta-Glucuronidase HPH: Hygromycin B phosphotransferase Luc: Luciferase NptII: Kanamycin/Neomycin phosphotransferase Spiking Controls B-cell receptor protein Myosin heavy chain Myosin Reg. Light chain Insulin-like growth factor FLJ109 fis HSPC 120 Beta2 microglobulin Phosphoglycerate kinase Tyrosine phosphatase G10 homolog

Probe Selection

NSF Project ITEC GenBank Private Collection

Processed ESTs cDNAs Gene Assembly: Phrap Clusters Gene Assembly: Phrap TS Blast TC Blast First EST Re-array

PCR

Miniprep DNA

Disrupted whole cells

Wheat Endosperm ESTs: Test Array Set Download Processed ESTs Gene Assembly:Phrap Tentative Singlets (TS) Blast Tentative Contigs (TC) Blast First EST Re-array Mini-prep DNA Verify I.D. (re-sequencing) PCR amplify inserts

(Remove vector, ribosomal, ct and mt DNA)

Array Scanner

ScanArray 5000XL

(from Packard BioChip Technologies)

Five lasers (488 nm-633nm excitation range) Eleven emission filters (500 nm -700 nm) Sensitivity to < 0.1 molecule fluor/um2 Up to 5 um resolution

Cy3/Cy5 Spectra

GeneChip Expression Analysis Process

Biotinylated RNA target from experimental sample

GeneChip expression

analysis probe array

Each probe cell contains millions of copies of a specific oligonucleotide probe

Image of hybridized probe array

Streptavidinphycoerythrin conjugate

GeneChip Expression Array Design

mRNA DNA probe pairs

Perfect Match Oligo Mismatch Oligo

Reference Sequence

Fluorescence Intensity Image

Perfect match probe cells

Mismatch probe cells

Lipshutz, RJ, Fodor, SPA, Gingeras, TR & DJ Lockhart, Nature Genetics Supplement, Vol 21, Jan 1999, pp 20-24

Light directed oligonucleotide synthesis

Lipshutz, R.J., Fodor, S.P.A., Gingeras, T.R. & D. LockhartNature Genetics Supl., Vol 21, Jan 1999, pp. 20-24

Applied Genomics Exploiting the human genome

· · · · · Molecular diagnostics of cancer SNPs and Personal pills Pharmacogenomics and new drugs Structural genomics and new targets and many more

SNP - single nucleotide polymorphism

· Variations in the gene sequence, resulting in the amino acid change in the protein, which results in the altered function

· Genotyping of oncogenes - cancer causing or permitting genes · Identifying and genotyping drug response gene variants

Genotyping Arrays

Allele A Allele B Genotype B/B Mismatch Perfect Match Perfect Match Mismatch A/A

A/B

120,000 probes for 3,000 biallelic loci

Biochip

· Microarrays · Microfluidics - "Lab on the Chip"

Saeks, J, GEN, Vol 21, May 15, 2001

On-Line Resources

· Affymetrix: http://www.affymetrix.com · Stanford: http://genome-www.stanford.edu/ · Silicon Genetics:

http:www.signetics.com/GeneSpring/Overview.htm

· Brown Lab, Stanford University: http://cmgm.stanford.edu/pbrown/explore · MicroArray Project, NIH: Image of hybridized probe array

http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/dbase.html

On-Line Resources

Affymetrix: http://www.affymetrix.com Stanford: http://genome-www.stanford.edu/ Silicon Genetics: http:www.signetics.com/GeneSpring/Overview.htm Brown Lab, Stanford University: http://cmgm.stanford.edu/pbrown/explore MicroArray Project, NIH:

http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/dbase.html

Course Evaluations (cont'd)

Satisfaction with the Course

The End

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