powerpoint presentationvgn/outreach/hybridizatio… · ppt file · web view · 2008-08-21title:...
TRANSCRIPT
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GeneChip Hybridization
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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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The hybridization oven
Target binds to the Probes
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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 [We hope]
Types: DNA to DNA DNA to RNA RNA to RNA PNA to DNA
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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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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]
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High Stringency vs. Low Stringency
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The fluidics station
Staining the biotinylated cRNAAn 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
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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
What is this shift called?
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The Scanned Yeast Array
220,000 probes
6,400 genes
11 um features
25 bp Sense DNA Oligo’s