Overview of Hybridization, Stringency, and Genechip Processing

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Overview of Hybridization, Stringency, and Genechip Processing

Presentation transcript:

Overview of Hybridization, Stringency, and Genechip Processing

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 Denature 99C 10 minutes Inject into GeneChip

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

Hybridization Optimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence Types: DNA to DNA DNA to RNA RNA to RNA LNA to DNA PNA to DNA PNA LNA

Stringency Stringency prevents: 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

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

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

Processing the Yeast Genechip

Three Components to the Affymetrix GeneChip System Hybridization oven -for hybridization of the target to the chip The Fluidic Station- for staining GS 3000 Scanner- for high resolution laser scanning of the stained chip

Staining the biotinylated fcRNA 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

Steps in the Staining Protocol Rinse away unhybridized FcRNA target Stain with Streptavidin PE [SAPE] Grand Total MW (Minimum) 292,800 150,244 735,844 Da WOW!!! Stain with Biotinylated IgG anti-SAPE antibody Stain AGAIN with Streptavidin PE [SAPE] Rinse throughly

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 Stoke shift Emission Excitation Wavelength

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

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

SMC-2007-GeneChip Image Data

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

QC Report From Genechip Actin Housekeeping Control 3’-5’ Ratio TATA BP

How well do the sample types correlate ?