overview of hybridization, stringency, and genechip processing
DESCRIPTION
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 Oligo1.7 ul - PowerPoint PPT PresentationTRANSCRIPT
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Overview of Hybridization, Stringency, and Genechip Processing
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The following hybridization mix is prepared for each sample
Fragmented cRNA 5ug 10 ul Control B2 Oligo 1.7 ul20x Eukaryotic Control mix [bio B, bio C, bio D, Cre] 5 ul Herring Sperm DNA [10mg/ml] 1 ul Acetyleted BSA [50mg/ml] 1 ulDMSO 10 ul2x Hybridization Buffer 50 ulWater 22.3 ul
Denature 99C
10 minutes
Inject into
GeneChip
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Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond
Targets:Antisense biotinylated cRNA
RNA-DNA Hybridization
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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 RNALNA to DNA PNA to DNA
PNA LNA
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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 formationDisassociation of strands
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Intrinsic factors
GC rich nucleic acid more stable because of triple H-bond
Degree of complementarity
Factors Influencing Stringency
Extrinsic factors
Experimentally introduced
TemperatureSalt concentration- NaCl, Na citrate, morpholinoethanesulfonic acidPresence of denaturing agents (e.g., formamide)Presence of high molecular weight polymers (e.g., dextran sulfate)Shear forcesMolecular tagging
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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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Processing the Yeast Genechip
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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)
292,800
150,244
292,800
735,844 Da
WOW!!!
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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
Excitation Wavelength
Emission
Stoke shift
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The Scanned Array
500,000 probe features
24,000 genes
18 um features
25 bp Sense DNA Oligo’s
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Microarray Images and QC
-Good for seeing visual defects-Examining Borders, Chip ID, Controls
Why do we look at this image?
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Castleton State College-GeneChip Image Data
csc 1 csc 2 csc4
csc 7 csc 8
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QC Report
-Check 3’ to 5’ ratios of housekeeping genesWhy do we look at the QC report?
-Scaling factor-Spike in control signal-Percent present
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GAPDH Control 3’-5’ Ratio
QC Report From Genechip
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How well do the sample types correlate ?