This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Expression monitoring by hybridization to high density oligonucleotide arrays

In order to obtain an estimate of the overall level of correlation between mRNA and protein abundances for a well-characterized pharmaceutically relevant biological system, we have analyzed human liver by quantitative two-dimensional electrophoresis (for protein abundances) and by Transcript Image methodology (for mRNA abundances). Incyte's LifeSeq database was searched for expressed sequence tag (EST) sequences corresponding to a series of 23 proteins identified on 2-D maps in the Large Scale Biology (LSB) Molecular Anatomy database, resulting in estimated abundances for 19 messages (4 were undetected) among 7926 liver clones sequenced. A correlation coefficient of 0.48 was obtained between the mRNA and protein abundances determined by the two approaches, suggesting that post-transcriptional regulation of gene expression is a frequent phenomenon in higher organisms. A comparison with published data (Kawamoto, S., et al., Gene 1996, 174, 151-158) on the abundances of liver mRNAs for plasma proteins (secreted by the liver) suggests that higher abundance messages are strongly enriched in secreted sequences. Our data confirms this: of the 50 most abundant liver mRNAs, 29 coded for secreted proteins, while none of the 50 most abundant proteins appeared to be secreted products (although four plasma and red blood cell proteins were present in this group as contaminants from tissue blood).

A comparison of selected mRNA and protein abundances in human liver

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

Very large scale immobilized polymer synthesis

The present invention provides method and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Method of detecting nucleic acids

A method and device for forming large arrays of polymers on a substrate ( 401 ). According to a preferred aspect of the invention, the substrate is contacted by a channel block ( 407 ) having channels ( 409 ) therein. Selected reagents are delivered through the channels, the substrate is rotated by a rotating stage ( 403 ), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

Combinatorial strategies for polymer synthesis

A computer-based scanning and image-processing system has been developed to quantitate the relative level of expression of each of 4000 cloned complementary DNA sequences in small biopsies routinely removed from the mucosa of normal and neoplastic human large intestine. Individuals have been studied from well-defined population groups in which colonic epithelial cells have progressed to increasingly advanced stages of neoplastic transformation. Comparison of normal colonic mucosa to colonic carcinomas demonstrated alterations in expression of approximately 7% of the cloned sequences; fewer changes were found between benign colonic adenomas and either normal colonic mucosa or carcinomas. A subset of the sequences which change in expression during progression from normal mucosa, to adenoma, to carcinoma showed complementary changes when colon carcinoma cells were induced to differentiate in vitro with sodium butyrate; quantitative correlations between in vivo and in vitro results were highly significant.

Comparison of normal colonic mucosa with mucosa from patients with the autosomal dominant disease familial polyposis revealed more extensive alterations in gene expression involving approximately 25% of the clones screened. Flat colonic mucosa in familial polyposis is therefore markedly aberrant and may be highly dedifferentiated, suggesting several possible mechanisms for the very high incidence of cancer that develops in this epithelium.

https://cancerres.aacrjournals.org/content/canres/47/22/6017.full.pdf

Leigh Anderson

Papers

Expression of Cloned Sequences in Biopsies of Human Colonic Tissue and in Colonic Carcinoma Cells Induced to Differentiate in Vitro