genechip ® probe arrays millions of copies of a specific oligonucleotide probe image of hybridized...

GeneChip® Probe Arrays

Millions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe

Image of Hybridized Probe ArrayImage of Hybridized Probe Array

>200,000 different>200,000 differentcomplementary probes complementary probes

Single stranded, Single stranded, labeled RNA targetlabeled RNA target

Oligonucleotide probeOligonucleotide probe

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*GeneChipGeneChip Probe ArrayProbe Array

Hybridized Probe CellHybridized Probe Cell

Courtesy of: Mike Lelivelt

Damla D. BilginUniv. of Illinois, Urbana

Photolithographic Synthesis

LampLamp

MaskMask ChipChip

Courtesy of: Mike LeliveltDamla D. BilginUniv. of Illinois, Urbana

Affymetrix Soybean Genome Array

The GeneChip® Soybean Genome Array can be used to study gene expression of over 37,500 soybean (Glycine max) transcripts.

The array includes probe sets to detect approximately 15,800 transcripts for Phytophthora sojae as well as 7,500 Heterodera glycines (cyst nematode pathogen) transcripts.


GeneChip® Expression Array Design

GeneGeneSequenceSequence

Probes designed to be Probes designed to be Perfect MatchPerfect Match

Probes designed to be Probes designed to be MismatchMismatch

3’ biased multiple 3’ biased multiple oligo probesoligo probes

UTR

5´5´ 3´3´


Synthesis of Ordered Oligonucleotide Arrays

O O O O O

Light(deprotection)

HO HO O O O T T O O O

T T C C O

Light(deprotection)

T T O O O

C A T A TA G C T GT T C C G

MaskMask

SubstrateSubstrate

MaskMask

SubstrateSubstrate

T –T –

C –C –REPEATREPEAT


GeneChip Probes (Affymetrix)

• 25-mer oligonucleotides synthesized directly on the glass

• 11-20 probe pairs are selected among all possible 25-mers to represent each transcript

• Perfect Match/Mismatch probe pair strategy


Probe Set 1

probe cell

probe pair

Probe Array (chip)

Probe Set 2

Probe Set 3


Probe – a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of the same probe molecules.

The intensity of each cell is an average of each of its scanned pixels.

Probe Cell20 - 50 micrometers

Pixel3 – 24 um


PM

MM

Probe PairEach pair represents a different subsequence of the gene

PMMM

MM

Probe Cell

Probe Set


keck.med.yale.edu Damla D. BilginUniv. of Illinois, Urbana

DefinitionsProbe – a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of probe molecules.Probe Array – a collection of probes sets.Probe Set – a set of probes designed to detect one transcript. 16-20 probe pairs. A 20 probe pair set is made up of 20 PM and 20 MM for a total of 40 probe cells.Probe Pair – Two probe cells, a PM and its corresponding MM.Perfect Match (PM) – probes that are designed to be complementary to the reference sequence.MisMatch (MM) – probes that are designed to be complementary to the reference sequence except for 1 base.Target – sequence from your sample.


Procedures for Target Preparation

cDNAcDNAFragmentFragment(heat, Mg(heat, Mg2+2+))

LL LL LL LL

Wash & StainWash & Stain

ScanScan

HybridizeHybridize

(16 hours)(16 hours)

Labeled transcriptLabeled transcript

Poly (A)Poly (A)++//TotalTotal RNARNA

AAAAAAAA

IVTIVT

(Biotin-UTP(Biotin-UTPBiotin-CTP)Biotin-CTP)

Labeled fragmentsLabeled fragments

LL LL

LL

LL

CellsCells


Argon laser488nm

570nm

Scanner based on epifluorescence confocal microscopy


Hybridization oven

Fluid station

Scanner


Affymetrix Core Facility at W.M. Keck Center at Univ. of Illinois at Urbana-Champaign


Useful web sites

http://www.affymetrix.com/products/arrays/index.affx

http://www.biotech.uiuc.edu/centers/Keck/Functional_genomics/

http://www.iobion.com/support/support_GT_training.html

http://www.cropsci.uiuc.edu/faculty/clough/

genechip ® probe arrays millions of copies of a specific oligonucleotide probe image of hybridized...

Documents

probe cells

probe pairs

probe cell20

probe pair set

millions of probe molecules

set of probes

mike leliveltdamla

target sequence