GeneChip Hybridization

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

The hybridization oven

Target binds to the Probes

Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond

Targets:Antisense biotinylated cRNA

RNA-DNA Hybridization

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 formationDisassociation of strands

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

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]

High Stringency vs. Low Stringency

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

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!!!

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

Excitation Wavelength

Emission

What is this shift called?

The Scanned Yeast Array

220,000 probes

6,400 genes

11 um features

25 bp Sense DNA Oligo’s