Nucleic acid analysis techniques

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 ...

AI Technical Summary

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 varies with the particular subsequence (region) of a gene being probed it is difficult to obtain an accurate and reliable measure of gene expression with one, or even a few, probes to the target gene.

Other methods, such as subtractive hybridization, do not require prior sequence knowledge, but also do not directly provide sequence information regarding differentially expressed nucleic acids.

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