GeneChip Hybridization

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GeneChip Hybridization
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The following hybridization mix is prepared for
each sample
Fragmented cRNA 5ug 10 ul
Control B2 Oligo 1.7 ul 20x Eukaryotic Control
mix bio B, bio C, bio D, Cre 5 ul Herring
Sperm DNA 10mg/ml 1 ul Acetyleted BSA
50mg/ml 1 ul DMSO 10 ul 2x Hybridization
Buffer 50 ul Water 22.3 ul
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The hybridization oven

• Target binds to the Probes

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RNA-DNA Hybridization
Targets Antisense biotinylated cRNA
Probe sets The DNA oligo probe is attached to
the GeneChip via a silane bond
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HybridizationOptimized Hybridization is the
process of single stranded nucleic acids binding
to another strand with identically complement
sequence We hope Types DNA to DNA
DNA to RNA RNA to RNA PNA to DNA
  
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Stringency   Stringency is a condition that
causes a change in the local hybridization
environment and interferes with the binding
kinetics Stringency prevents  . Binding
of non-complementary strands Self hybridization
hairpin formation Disassociation of strands
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Factors Influencing Stringency
Intrinsic factors  GC rich nucleic acid more
stable because of triple H-bond  Degree of
complementarity
Extrinsic factors Experimentally
introduced Temperature Salt concentration-
NaCl, Na citrate, morpholinoethanesulfonic
acid Presence of denaturing agents (e.g.,
formamide) Presence of high molecular weight
polymers (e.g., dextran sulfate) Shear
forces Molecular tagging
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This is different then PCR, because increasing
salt concentration increases stringencyThis is
because of the enzyme activity of taq polymerase
and Molecular interference
Stringency In Microarray Hybridization
High stringency is obtained by Low salt or
buffer concentration High temperature
Low stringency is obtained by Lowering the
temperature of hybridization Increasing salt
concentration to a point
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High Stringency vs. Low Stringency
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The fluidics station

• Staining the biotinylated cRNA
• An automated system to stain the target using
  streptavidin-phycoerythrin SAPE, a biotinylated
  anti-SAPE antibody, and SAPE again
• high and low stringency buffers are used

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Steps in the Staining Protocol
Rinse away unhybridized FcRNA target
Stain with Streptavidin PE SAPE
Grand Total MW (Minimum) 292,800 150,244 292,800
735,844 Da WOW!!!
Stain with Biotinylated IgG anti-SAPE antibody
Stain AGAIN with Streptavidin PE SAPE
Rinse throughly
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The Staining Chemistry for Affymetrix Genechip
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Scanning the Yeast 2.0 GeneChip with the
GS3000-Nd-YAG laser 532nm-2.5 uM resolution
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Fluorescent Spectrum of Phycoerythrin
Emission
Excitation Wavelength
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The Scanned Yeast Array220,000 probes6,400
genes11 um features25 bp Sense DNA Oligos