Expression monitoring by hybridization to high-density oligonucleotide arrays.
The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.
Lockhart
D J
DJ
Affymetrix, Santa Clara, CA 95051, USA. david_lockhart@affymetrix.com
Dong
H
H
Byrne
M C
MC
Follettie
M T
MT
Gallo
M V
MV
Chee
M S
MS
Mittmann
M
M
Wang
C
C
Kobayashi
M
M
Horton
H
H
Brown
E L
EL
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002874:Chromosome Mapping; D016207:Cytokines / Q000235:genetics; D017931:DNA Primers / Q000138:chemical synthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007476:Ionophores; D051379:Mice; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011061:Poly A; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D007476:Ionophores; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D013755:Tetradecanoylphorbol Acetate