GeneChip (Affymetrix)

Introduction

Micro-arrays of short oligonucleotides 25 base pairs directly synthesized onto the surface of the chip.

Methods

Array size ranges from 5000-15,000.

Standard procedure requires a recommended 5 ug of total RNA.

GeneChip probe arrays are manufactured using technology that combines photolithography & combinatorial chemistry. Thousands of different oligonucleotide probes are synthesized on each array. Each oligonucleotide is located in a specific area on the array called a probe cell. Each probe cell contains > 10E06 of copies of a given oligonucleotide or probe. For each transcript, 22-25 oligonucleotides of length 25 representing sequences & single base-pair mismatch sequences distributed over 600 base pairs in the 3' region of the transcript. These 22-25 oligo- nucleotides are distributed throughout the chip.

Probe arrays are manufactured in a series of cycles. Initially, a glass substrate is coated with linkers containing photolabile protecting groups. Then, a mask is applied that exposes selected portions of the probe array to ultraviolet light. Illumination removes the photolabile protecting groups enabling selective nucleoside phosphoramidite addition only at the previously exposed sites. Next, a different mask is applied & the cycle of illumination & chemical coupling is repeated. Through this cycle, a specific set of oligonucleotide probes is synthesized with each probe type in a specific location. The completed probe arrays are packaged into cartridges.

For eukaryotic samples, double-stranded cDNA is synthesized from total RNA or purified poly-A mRNA isolated from tissue or cells. An in vitro transcription (IVT) reaction is then performed to produce biotin-labeled cRNA from the cDNA. The cRNA is fragmented before hybridization.

A hybridization cocktail is prepared, including the biotin- labeled cRNA fragments, probe array controls, BSA, & herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation. The hybridized probe array is stained with streptavidin phycoerythrin conjugate & scanned by the GeneArray Scanner at the excitation wavelength of 488 nm. The amount of light emitted at 570 nm is proportional to the bound target at each location on the probe array.

More general terms

• DNA microarray (gene chip)

Additional terms

• GeneChip array data