Gene. Chip Hybridization
The following hybridization mix is prepared for each sample Fragmented c. RNA 5 ug 10 ul Control B 2 Oligo 1. 7 ul 20 x Eukaryotic Control mix [bio B, bio C, bio D, Cre] Herring Sperm DNA [10 mg/ml] 1 ul Acetyleted BSA [50 mg/ml] 1 ul DMSO 10 ul 2 x Hybridization Buffer Water 22. 3 ul 50 ul Denature 99 C Inject into 10 minutes Gene. Chip
The hybridization oven Target binds to the Probes
RNA-DNA Hybridization Targets: Antisense biotinylated c. RNA Probe sets: The DNA oligo probe is attached to the Gene. Chip via a silane bond
Hybridization Optimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence [We hope] Types: DNA to RNA to RNA PNA to DNA
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- Na. Cl, 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] This is different then PCR, because increasing salt concentration increases stringency This is because of the enzyme activity of taq polymerase and Molecular interference
High Stringency vs. Low Stringency
The fluidics station Staining the biotinylated c. RNA 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 Fc. RNA target Stain with Streptavidin PE [SAPE] Stain with Biotinylated Ig. G anti-SAPE antibody Stain AGAIN with Streptavidin PE [SAPE] Grand Total MW (Minimum) 292, 800 150, 244 Rinse throughly 292, 800 735, 844 Da WOW!!!
The Staining Chemistry for Affymetrix Genechip
Scanning the Yeast 2. 0 Gene. Chip with the GS 3000 -Nd-YAG laser 532 nm -2. 5 u. M resolution
Fluorescent Spectrum of Phycoerythrin What is this shift called? Emission Excitation Wavelength
The Scanned Yeast Array 220, 000 probes 6, 400 genes 11 um features 25 bp Sense DNA Oligo’s
Скачать презентацию Gene Chip Hybridization The following hybridization mix
1a3f3c4c0b2ed079b3875df119e0de94.ppt