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GeneChip Hybridization

GeneChip Hybridization. Denature 99C 10 minutes. Inject into GeneChip. 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

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GeneChip Hybridization

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  1. GeneChip Hybridization

  2. Denature 99C 10 minutes Inject into GeneChip 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 Water22.3 ul

  3. The hybridization oven Target binds to the Probes

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

  5. 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

  6. 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

  7. Factors Influencing Stringency Intrinsic factorsGC 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

  8. Stringency In Microarray Hybridization This is different then PCR, because increasing salt concentration increases stringencyThis is because of the enzyme activity of taq polymerase and Molecular interference 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]

  9. High Stringency vs. Low Stringency

  10. 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

  11. 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

  12. The Staining Chemistry for Affymetrix Genechip

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

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

  15. The Scanned Yeast Array220,000 probes6,400 genes11 um features25 bp Sense DNA Oligo’s

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