Nucleic acid analysis techniques

a technology of nucleic acid and analysis techniques, applied in the field of nucleic acid analysis techniques, can solve the problems of difficult or impossible to distinguish two or more gene products of approximately the same molecular weight, difficult to obtain an accurate and reliable measure of gene expression with one, or even a few, probes to the target gene, etc., and achieves the effect of simple application and rapidity

Inactive Publication Date: 2005-07-21
AFFYMETRIX INC
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Benefits of technology

[0008] The present invention, in one embodiment, provides methods of monitoring the expression of a multiplicity of preselected genes (referred to herein as “expression monitoring”). In another embodiment this invention provides a way of identifying differences in the compositions of two or more nucleic acid (e.g., RNA or DNA) samples. Where the nucleic acid abundances reflect expression levels in biological samples from which the s

Problems solved by technology

The use of “traditional” hybridization protocols for monitoring or quantifying gene expression is problematic.
For example two or more gene products of approximately the same molecular weight will prove difficult or impossible to distinguish in a Northern blot because they are not readily separated by electrophoretic methods.
Similarly, as hybridization efficiency and cross-reactivity vari

Method used

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Examples

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example 1

First Generation Oligonucleotide Arrays Designed to Measure mRNA Levels for a Small Number of Murine Cytokines

A) Preparation of Labeled RNA.

[0479] 1) From Each of the Preselected Genes.

[0480] Fourteen genes (IL-2, IL-3, Il-4, IL-6, Il-10, IL-12p40, GM-CSF, IFN-γ, TNF-α, CTLA8, β-actin, GAPDH, IL-11 receptor, and Bio B) were each cloned into the p Bluescript II KS (+) phagemid (Stratagene, La Jolla, Calif., USA). The orientation of the insert was such that T3 RNA polymerase gave sense transcripts and T7 polymerase gave antisense RNA.

[0481] Labeled ribonucleotides in an in vitro transcription (IVT) reaction. Either biotin- or fluorescein-labeled UTP and CTP (1:3 labeled to unlabeled) plus unlabeled ATP and GTP were used for the reaction with 2500 units of T7 RNA polymerase (Epicentre Technologies, Madison, Wis., USA). In vitro transcription was done with cut templates in a manner like that described by Melton et al., Nucleic Acids Research, 12: 7035-7056 (1984). A typical in vitr...

example 2

T Cell Induction Experiments Measuring Cytokine mRNAs as a Function of Time Following Stimulation

[0525] The high density arrays of this invention were next used to monitor cytokine MRNA levels in murine T cells at different times following a biochemical stimulus. Cells from the murine T helper cell line (2D6) were treated with the phorbol ester 4-phorbol-12-myristate 13-acetate (PMA) and a calcium ionophore. Poly (A)+ MRNA was then isolated at 0, 2, 6 and 24 hours after stimulation. Isolated mRNA (approximately 1 μg) was converted to labeled antisense RNA using a procedure that combines a double-stranded cDNA synthesis step with a subsequent in vitro transcription reaction. This RNA synthesis and labeling procedure amplifies the entire mRNA population by 20 to 50-fold in an apparently unbiased and reproducible fashion (Table 2).

[0526] The labeled antisense T-cell RNA from the four time points was then hybridized to DNA probe arrays for 2 and 22 hours. A large increase in the γ-int...

example 3

Higher-Density Arrays Containing 65,000 Probes for Over 100 Murine Genes

[0528]FIG. 5 shows an array that contains over 65,000 different oligonucleotide probes (50 μm feature size) following hybridization with an entire murine B cell RNA population. Arrays of this complexity were read at a resolution of 7.5 μm in less than fifteen minutes. The array contains probes for 118 genes including 12 murine genes represented on the simpler array described above, 35 U.S.C. § 1020 additional murine genes, three bacterial genes and one phage gene. There are approximately 300 probe pairs per gene, with the probes chosen using the selection rules described herein. The probes were chosen from the 600 bases of sequence at the 3′ end of the translated region of each gene. A total of 21 murine RNAs were unambiguously detected in the B cell RNA population, at levels ranging from approximately 1:300,000 to 1:100.

[0529] Labeled RNA samples from the T cell induction experiments (FIG. 4) were hybridized ...

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Abstract

The present invention provides a simplified method for identifying differences in nucleic acid abundances (e.g., expression levels) between two or more samples. The methods involve providing an array containing a large number (e.g. greater than 1,000) of arbitrarily selected different oligonucleotide probes where the sequence and location of each different probe is known. Nucleic acid samples (e.g. mRNA) from two or more samples are hybridized to the probe arrays and the pattern of hybridization is detected. Differences in the hybridization patterns between the samples indicates differences in expression of various genes between those samples. This invention also provides a method of end-labeling a nucleic acid. In one embodiment, the method involves providing a nucleic acid, providing a labeled oligonucleotide and then enzymatically ligating the oligonucleotide to the nucleic acid. Thus, for example, where the nucleic acid is an RNA, a labeled oligoribonucleotide can be ligated using an RNA ligase. In another embodiment, the end labeling can be accomplished by providing a nucleic acid, providing labeled nucleoside triphosphates, and attaching the nucleoside triphosphates to the nucleic acid using a terminal transferase.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This is a continuation-in-part of U.S. Ser. No. 60 / 010,471 filed on Jan. 23, 1996 and a continuation-in-part of provisional patent application for “Labeling of Nucleic Acids” naming Lockhart, Cronin, Lee, Tran, Matsuzaki, McGall and Barone as inventors, filed on Jan. 9, 1997, both of which are herein incorporated by reference for all purposes.BACKGROUND OF THE INVENTION [0002] A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the xerographic reproduction by anyone of the patent document or the patent disclosure in exactly the form it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. [0003] Many disease states are characterized by differences in the expression levels of various genes either through changes in the copy number of the genetic DNA or through changes in ...

Claims

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Application Information

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IPC IPC(8): C07B61/00C07H19/052C07H19/12C07H21/00C12Q1/00C12Q1/68G16B25/20G16B30/10
CPCC07H19/052C07H19/12G06F19/22G06F19/20C40B40/00C12Q2600/156C12Q1/6837C12Q1/6809C07H21/00C12Q2525/161C12Q2565/501C12Q2561/125G16B25/00G16B30/00G16B30/10G16B25/20
Inventor LOCKHART, DAVIDCHEE, MARKGUNDERSON, KEVINCHAOQIANG, LAIWODICKA, LISACRONIN, MAUREENLEE, DANNYTRAN, HUUMATSUZAKI, HAJIMEMCGALL, GLENNBARONE, ANTHONY
Owner AFFYMETRIX INC
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