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Overview of Hybridization, Stringency, and Genechip Processing

Overview of Hybridization, Stringency, and Genechip Processing. 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

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Overview of Hybridization, Stringency, and Genechip Processing

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  1. Overview of Hybridization, Stringency, and Genechip Processing

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

  4. HybridizationOptimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequenceTypes: DNA to DNA DNA to RNA RNA to RNA LNA to DNA PNA to DNA PNA LNA

  5. 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

  6. 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

  7. 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]

  8. High Stringency vs. Low Stringency

  9. Processing the Yeast Genechip

  10. Three Components to the Affymetrix GeneChip SystemHybridization oven -for hybridization of the target to the chipThe Fluidic Station- for stainingGS 3000 Scanner- for high resolution laser scanning of the stained chip

  11. The Fluidics Station Staining the biotinylated fcRNA 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

  12. 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

  13. The Staining Chemistry for Affymetrix Genechip

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

  15. Stoke shift Fluorescent Spectrum of Phycoerythrin Emission Excitation Wavelength

  16. The Scanned Array500,000 probe features24,000 genes18 um features25 bp Sense DNA Oligo’s

  17. Microarray Images and QC Why do we look at this image? -Good for seeing visual defects -Examining Borders, Chip ID, Controls

  18. Marlboro College-GeneChip Image Data

  19. QC Report Why do we look at the QC report? • Check 3’ to 5’ ratios of housekeeping genes -Scaling factor -Spike in control signal -Percent present

  20. QC Report From Genechip GAPDH Control 3’-5’ Ratio

  21. How well do the sample types correlate ?

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