GeneChip Hybridization. The following hybridization mix is prepared for each sample Fragmented cRNA 5ug 10 ul Control B2 Oligo1.7 ul 20x Eukaryotic Control.

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Presentation transcript:

GeneChip Hybridization

The following hybridization mix is prepared for each sample Fragmented cRNA 5ug 10 ul Control B2 Oligo1.7 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 DMSO10 ul 2x Hybridization Buffer 50 ul Water22.3 ul Denature 99C 10 minutes Inject into GeneChip

The hybridization oven Target binds to the Probes

Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond Targets: Antisense biotinylated cRNA RNA-DNA Hybridization

Hybridization Optimized 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

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

Intrinsic factors GC rich nucleic acid more stable because of triple H-bond Degree of complementarity Factors Influencing Stringency 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

This is different then PCR, because increasing salt concentration increases stringency This 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 ]

High Stringency vs. Low Stringency

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

Steps in the Staining Protocol Rinse away unhybridized FcRNA target Stain with Streptavidin PE [SAPE] Stain with Biotinylated IgG anti-SAPE antibody Stain AGAIN with Streptavidin PE [SAPE] Rinse throughly Grand Total MW (Minimum) 292, , , ,844 Da WOW!!!

The Staining Chemistry for Affymetrix Genechip

Scanning the Yeast 2.0 GeneChip with the GS3000 -Nd-YAG laser 532nm -2.5 uM resolution

Fluorescent Spectrum of Phycoerythrin Excitation Wavelength Emission What is this shift called?

The Scanned Yeast Array 220,000 probes 6,400 genes 11 um features 25 bp Sense DNA Oligo’s