GeneChip Hybridization
The following hybridization mix is prepared for each sample
Fragmented cRNA
5ug
Control B2 Oligo
20x Eukaryotic Control mix [bio B, bio C, bio D, Cre]
Herring Sperm DNA [10mg/ml]
Acetyleted BSA [50mg/ml]
DMSO
2x Hybridization Buffer
Water
10 ul
1.7 ul
5 ul
1 ul
1 ul
10 ul
50 ul
22.3 ul
Denature 99C
Inject into
10 minutes
GeneChip
The hybridization oven
Target binds to the Probes
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 [We hope]
Types:
DNA to DNA
DNA to RNA
RNA to RNA
PNA to DNA
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
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]
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 cRNA
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]
Stain with Biotinylated IgG anti-SAPE antibody
Grand Total MW
(Minimum)
Stain AGAIN with Streptavidin PE [SAPE]
292,800
150,244
Rinse throughly
292,800
735,844 Da
WOW!!!
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
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