Solid phase selection of differentially expressed genes
First Claim
1. A method of analyzing relative gene expression in a plurality of different cells or tissues, the method comprising the steps of:
- providing a reference DNA population of DNA sequences attached to septerate solid phase supports in clonal subpopulations;
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; and
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 polynucleotides from the same gene of the different cells or tissues hybeidize with the same complementary sequence of the reference DNA population and such that such 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
sorting each solid phase support according to a relative optical signal generated by the light-generating labels of the duplexes attached thereto.
3 Assignments
0 Petitions
Accused Products
Abstract
The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluoresent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.
102 Citations
16 Claims
-
1. A method of analyzing relative gene expression in a plurality of different cells or tissues, the method comprising the steps of:
-
providing a reference DNA population of DNA sequences attached to septerate solid phase supports in clonal subpopulations;
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; and
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 polynucleotides from the same gene of the different cells or tissues hybeidize with the same complementary sequence of the reference DNA population and such that such 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
sorting each solid phase support according to a relative optical signal generated by the light-generating labels of the duplexes attached thereto. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
accumulating in a seperate vessel each said solid phase support having said relative optical signal with a value within one or more predetermined ranges of values corresponding to a difference in gene expression among said plurality of different cells or tissues; and
identifying said polynucleotides on each of said solid supports by determining a nucleotide sequence of a portion of each of said polynucleotides.
-
-
6. The method of claim 5 wherein said relative optical signal is a ratio of fluorescence intensities and wherein said plurality of different cells or tissues is two.
-
7. The method of claim 6 wherein said portion of said polynucleotides is a sequence of at least ten nucleotides.
-
8. The method of claim 6 wherein said step of identifying includes simultaneous sequencing of at least ten thousand of said polynucleotides by massively parallel signature sequencing.
-
9. A method of isolating polynucleotides derived from genes differentially expressed in a plurality of different cells or tissues, the method comprising the steps of:
-
providing a reference DNA population of DNA sequences attached to separate microparticles in clonal subpopulations;
providing a population of polynucleotides derived from genes expressed in each of the plurality of different cells or tissues, each polynucleotide having a light-generating label capable of generating an optical signal indicative of the cells or tissues from which it is derived;
competitively hybridizing the populations of polynucleotides of genes expressed in 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 from each of the different cells or tissues such that polynucleotides from the same gene of the different cells or tissues hybridize with the same complementary sequence of the reference DNA population and such that such polynucleotides are present in dupleses on each of the microparticles in ratios directly related to the relative expression of their corresponding genes in the different cells or tissues; and
isolating polynucleotides corresponding to genes differentially expressed in the different cells or tissues by sorting microparticles in accordance with the optical signals generated by the populations of polynucleotides hybridized thereto. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
-
Specification