Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-06-25
2002-02-05
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06344316
ABSTRACT:
BACKGROUND OF THE INVENTION
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.
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 levels of transcription (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) of particular genes. For example, losses and gains of genetic material play an important role in malignant transformation and progression. These gains and losses are thought to be “driven” by at least two kinds of genes. Oncogenes are positive regulators of tumorigenesis, while tumor suppressor genes are negative regulators of tumorigenesis (Marshall,
Cell,
64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991)). Therefore, one mechanism of activating unregulated growth is to increase the number of genes coding for oncogene proteins or to increase the level of expression of these oncogenes (e.g. in response to cellular or environmental changes), and another is to lose genetic material or to decrease the level of expression of genes that code for tumor suppressors. This model is supported by the losses and gains of genetic material associated with glioma progression (Mikkelson et al.
J. Cell. Biochem.
46: 3-8 (1991)). Thus, changes in the expression (transcription) levels of particular genes (e.g. oncogenes or tumor suppressors), serve as signposts for the presence and progression of various cancers.
Similarly, control of the cell cycle and cell development, as well as diseases are characterized by the variations in the transcription levels of particular genes. Thus, for example, a viral infection is often characterized by the elevated expression of genes of the particular virus. For example, outbreaks of Herpes simplex, Epstein-Barr virus infections (e.g. infectious mononucleosis), cytomegalovirus, Varicella-zoster virus infections, parvovirus infections, human papillomavirus infections, etc. are all characterized by elevated expression of various genes present in the respective virus. Detection of elevated expression levels of characteristic viral genes provides an effective diagnostic of the disease state. In particular, viruses such as herpes simplex, enter quiescent states for periods of time only to erupt in brief periods of rapid replication. Detection of expression levels of characteristic viral genes allows detection of such active proliferative (and presumably infective) states.
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.
The development of VLSIPS™ technology provided methods for synthesizing arrays of many different oligonucleotide probes that occupy a very small surface area. See U.S. Pat. No. 5,143,854 and PCT patent publication No. WO 90/15070. U.S. patent application Ser. No. 082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific nucleotide sequence.
Previous methods of measuring nucleic acid abundance differences or changes in the expression of various genes (e.g., differential diaplay, SAGE, cDNA sequencing, clone spotting, etc.) require assumptions about, or prior knowledge regarding the target sequences in order to design appropriate sequence-specific probes. 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.
SUMMARY OF THE INVENTION
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 samples are derived, the invention provides a method for identifying differences in expression profiles between two or more samples. These “generic difference screening methods” are rapid, simple to apply, require no a priori assumptions regarding the particular sequences whose expression may differ between the two samples, and provide direct sequence information regarding the nucleic acids whose abundances differ between the samples.
In one embodiment, this invention provides a method of identifying differences in nucleic acid levels between two or more nucleic acid samples. The method involves the steps of: (a) providing one or more oligonucleotide arrays said arrays comprising probe oligonucleotides attached to a surface; (b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays that are complementary to said nucleic acids or subsequences thereof; (c) contacting said one or more arrays with a nucleic acid ligase; and (d) determining differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.
In another embodiment, the method of identifying differences in nucleic acid levels between two or more nucleic acid samples involves the steps of: (a) providing one or more oligonucleotide arrays comprising probe oligonucleotides wherein said probe oligonucleotides comprise a constant region and a variable region; (b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and said variable regions that are complementary to said nucleic acids or subsequences thereof; and (c) determining differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.
In yet another embodiment, the method of identifying differences in nucleic acid levels between two or more nucleic acid samples involves the steps of: (a) providing one or more high density oligonucleotide arrays; (b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays that are complementary to said nucleic acids or subsequences thereof; and (c) determining the differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.
In still yet another embodiment, the method of identifying differences in nucleic acid levels between two or more nucleic acid samples involves the steps of: (a) providing one or more oligonucleotide arrays each comprising probe oligonucleotides wherein said probe oligonucleotides are not chosen to hybridize to nucleic acids derived from particular preselected genes or mRNAs; (b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays t
Chaoqiang Lai
Chee Mark
Cronin Maureen T.
Gunderson Kevin
Lee Danny
Affymetrix Inc.
Houtteman Scott W.
Townsend and Townsend / and Crew LLP
LandOfFree
Nucleic acid analysis techniques does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid analysis techniques, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid analysis techniques will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2949609