文档介绍：Overview of Affymetrix GeneChip Technology
GeneChip Technology Affymetrix Inc
Miniaturized, high density arrays
Expression arrays 1,300,000 DNA oligos 1-cm by 1-cm
DNA mapping array 7,000,000 DNA oligo by cm
Manufacturing Process
Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry
Photolithographic Synthesis Manufacturing Process Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of pounds in precise locations
Lamp
Mask
Chip
Affymetrix Wafer and Chip Format
cm
20 - 50 µm
20 - 50 µm
one oligonucleotide
sequence per “pixel”
49 - 400 chips/wafer
up to ~ million features/chip
Selection of Expression Probes Set of oligos to be synthesized is defined from sense sequence of known genes and EST’s
Probes
Sequence
Perfect Match
Mismatch
Chip
5’
3’
Computer algorithms are used to design photolithographic masks for use in manufacturing
Each gene is represented on the probe array by multiple probe pairs
Each probe pair consists of a perfect match and a mismatch oligonucleotide
Overview: Creating Targets
Reverse Transcriptase
in vitro transcription
mRNA
cDNA
Fragmentation of cRNA
cRNA
GeneChip Hybridization
This is what we are doing in this class
mRNA cRNA
Fragmentation of biotinylated cRNA