Solid phase selection of differentially expressed genes

Details Solid phase selection of differentially expressed genes Solid phase selection of differentially expressed genes

1998-08-06

2001-07-24

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S005000, C536S024310, C536S024320, C536S025320

Reexamination Certificate

active

06265163

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to methods for identifiing differentially expressed genes, and more particularly, to a method of competitively hybridizing differentially expressed DNAs with reference DNA sequences cloned on solid phase supports to provide a differential expression library which can be physically manipulated, e.g. by fluorescence-activated flow sorting.
BACKGROUND
The desire to decode the human genome and to understand the genetic basis of disease and a host of other physiological states associated differential gene expression has been a key driving force in the development of improved methods for analyzing and sequencing DNA, Adams et al, Editors, Automated DNA Sequencing and Analysis (Academic Press, New York, 1994). Current genome sequencing projects use Sanger-based sequencing technologies, which enable the sequencing and assembly of a genomes in the size range of 2-4 megabases with about 24 man-months of effort, e.g. Fleischmann et al, Science, 269: 496-512 (1995). Such a genome is about 0.005 the size of the human genome, which is estimated to contain about 10
5
genes, 15% of which—or about 3 megabases-are active in any given tissue. The large numbers of expressed genes make it difficult to track changes in expression patterns by direct sequence analysis. More commonly, expression patterns are analyzed by lower resolution techniques, such as differential display, indexing, subtraction hybridization, or one of the numerous DNA fingerprinting techniques, e.g. Lingo et al, Science, 257: 967-971 (1992); Erlander et al, International patent application PCT/US94/13041; McClelland et al, U.S. Pat. No. 5,437,975; Unrau et al, Gene, 145: 163-169 (1994); Sagerstrom et al, Annu. Rev. Biochem. 66: 751-783 (1997); and the like. For the techniques that result in the isolation of a subset of DNA sequences, sequencing of randomly selected clones is typically carried out using conventional Sanger sequencing; thus, the scale of the analysis is limited.
Recently, several higher resolution techniques have been reported that attempt to provide direct sequence information for analyzing patterns of gene expression on a large scale: Schena et al, Science, 270: 467-469 (1995), and DeRisi et al, Science, 278: 680-686 (1997), report the hybridization of mRNAs to a collection of cDNAs arrayed on a glass slide; Velculescu et al, Science, 270: 484-486 (1995) report the excision and concatenation of short segments of sequence adjacent to type iHs restriction sites from members of a cDNA library, followed by Sanger sequencing of the concatenated segments to give a profile of sequences in the library; and Wodicka et al, Nature Biotechnology, 15: 1359-1367 (1997), report genome-wide expression monitoring of yeast under different growth conditions using high density oligonucleotide arrays containing hybridization sites for each of the more than 6000 genes of the organism. While these techniques represent tremendous progress in expression analysis, they still have drawbacks which limit their widespread application to many expression monitoring problems. For example, in both the techniques of Schena and Wodicka, the sequences being monitored must be known beforehand, and in the case of Wodicka preferably the entire complement of an organism's genes must be known. In the technique of Schena, there are significant problems in constructing arrays containing a substantial portion, e.g. ten thousand, or more, of genes whose expression may be relevant, as cDNAs of each gene are separately prepared an applied to an array and currently available arrays are typically not re-usable leading to standardization and quality control issues when multiple measurements over time are desired. In the technique Velculescu, even though the sequencing burden is reduced, as with any random sequencing approach, abundant non-differentially expressed genes are sequenced repeatedly—at the expense of obtaining expression information on differentially regulated genes, and it is not clear from the reported data whether the technique is capable of providing sample sizes sufficiently large to permit the reliable expression profiling of genes that are expressed very low levels, e.g. Kollner et al, Genomics, 23: 185-191 (1994).
In view of the above, it would be highly desirable if a technique were available for monitoring differential gene expression that had the capability of massively parallel analysis of all or a substantial fraction of expressed genes, but was free of the shortcomings of current techniques.
SUMMARY OF THE INVENTION
Accordingly, objects of our invention include, but are not limited to, providing a method for identifying and isolating differentially expressed genes or polymorphic genes; providing a method of identifying and isolating polynucleotides on the basis of labels that generate different optical signals; providing a method for profiling gene expression of large numbers of genes simulaneously or identifying large numbers of polymorpbic genes simultaneously; providing a method of identifying and separating genes in accordance with whether their expression is increased or decrease under any given conditions; and providing a method for massively parallel signature sequencing of large numbers of genes isolated according to their expression characteristics or according to whether they contain a polymorphic sequence.
Our invention accomplishes these and other objects by providing differently labeled populations of DNAs from cell or tissue sources whose gene expression is to be compared. In comparing gene expression, differently labeled DNAs of a plurality of populations are competitively hybridized with reference DNA cloned on solid phase supports. Preferably, the solid phase supports are microparticles which, after such competitive hybridization, provide a differential expression library which may be manipulated by fluorescence-activated cell sorting (FACS), or other sorting means responsive to optical signals generated by labeled DNA on the microparticles. Monitoring the relative signal intensity of the different labels on the microparticles permits quantification of the relative expression of particular genes in the different populations.
In one aspect of the invention, populations of microparticles having relative signal intensities of interest are isolated by FACS and the attached DNAs are sequenced to determine the identities of the differentially expressed genes.
Preferably, the method of the invention is carried out by the following steps: a) providing a reference DNA population of DNA sequences attached to separate solid phase supports in clonal subpopulations; b) providing a population of polynucleotides of expressed genes from each of the plurality of different cells or tissues, the polynucleotides of expressed genes from different cells or tissues having a different light-generating label and the polynucleotides of expressed genes from the same cells or tissues having the same light-generating label; c) competitively hybridizing the populations of polynucleotides of expressed genes from each of the plurality of different cells or tissues with the reference DNA population to form duplexes between the DNA sequences of the reference DNA population and polynucleotides of each of the different cells or tissues such that the polynucleotides are present in duplexes on each of the solid phase supports in ratios directly related to the relative expression of their corresponding genes in the different cells or tissues; and d) sorting each solid phase support according to a relative optical signal generated by the light-generating labels of the duplexes attached thereto. Preferably, the reference DNA population is derived from genes of the plurality of different cells or tissues being analyzed. As used herein, the phrase “polynucleotides of expressed genes” is meant to include any RNA produced by transcription, including in particular M/RNA, and DNA produced by reverse transcription of any RNA, including in particular cDNA produced by reverse transcription of M/RNA.
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