Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-02
2002-05-07
Fredman, Jeffrey (Department: 1643)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S320100, C536S023100, C536S024300
Reexamination Certificate
active
06383749
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is nucleic acid labeling, particularly labeling of nucleic acid targets for use in array based hybridization assays.
2. Background of the Invention
Nucleic acid arrays have become an increasingly important tool in the biotechnology industry and related fields. Nucleic acid arrays, in which a plurality of nucleic acids are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. One important use of nucleic acid arrays is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
In many currently employed array based gene expression analysis protocols, differences in mRNA levels between two samples are detected and related to the expression level of different genes in the compared samples. Detection of different mRNA levels typically involves the steps of generating a target nucleic acid population that is representative of the mRNA population of the test sample. In other words, a population of target nucleic acids is generated where the population is indicative of the different mRNA levels that are originally present in the sample. The target nucleic acid population may be DNA or RNA and may have the sequence of the initial mRNA or the complement thereof. Following generation, the population of target nucleic acids is hybridized to an array of probe nucleic acids stably associated with the surface of a solid support. Since the sequence and location of each probe is known, any resultant hybridization complexes that form on the array surface between target and probe can be used to identify those genes that are expressed in the cell from which the initial mRNA sample was obtained. The intensity of the individual signals can also be used to at least semi-quantitatively determine the expression level of the detected genes. Since the methods require detection of target/probe complexes on the array surface, the target nucleic acids are generally labeled so that they can be detected.
In many embodiments, the target nucleic acids are labeled during their generation step. In other words, the targets that are generated from the initial sample are labeled targets. A number of different protocols have been developed for producing populations of the labeled target nucleic acids from an initial source. Such methods include: (a) those based on the use of labeled primer; (b) those based on the use of one or more labeled nucleotides; and the like. While the above approaches are effective in many situations, they are not perfect. For example, the spectrum of fluorescent labels that may be employed in protocols where labeled targets are generated from labeled nucleotide analogs is limited, as not all fluorescently tagged nucleotide analogs can be processed by enzymes, e.g. polymerases, that are employed in the labeled target generation step.
As such, there is continued interest in the development of new protocols for producing labeled target nucleic acids. Of particular interest would be the development of a protocol which is suitable for producing fluorescently labeled target nucleic acids, in which the fluorescent label is covalently bound to the nucleic acid, where the protocol provides for the use of a broader range of fluorescent labels that can be used in current protocols where fluorescently tagged nucleotide analogs are employed in target generation.
Relevant Literature
Patents of interest include: U.S. Pat. Nos. 5,684,142; 5,286,486 and 5,241,060. Other references of interest include: Chu et al., Nuc. Acids Res. (July 1986) 14:5591-5603; Gebeyehu et al., Nuc. Acids Res. (1987) 15:4513; Griffor et al.., Plant Mol. Biol. (July 1991) 17:101-109; Langer et al., Proc. Nat'l Acad. Sci. U.S.A. (1981) 78:6633; and Pinkel et al., Proc. Nat'l Acad. Sci. USA (May 1986) 83:2934-8. See also: Methods in Molecule Biology 28: Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Isaac ed.)(Humana Press 1994); Hermanson, Bioconjugate Techniques (Academic Press, 1995); and Nonisotopic Probing, Blotting, and Sequencing (Kricka ed) Academic Press, 1995).
SUMMARY OF THE INVENTION
Methods and kits are provided for labeling nucleic acids, e.g. for use in array based hybridization assays. In the subject methods, a population of target nucleic acid is first generated from an initial nucleic acid source, e.g. mRNA, where each target nucleic acid in the population is characterized by having at least one reactive functionality that is not a moiety found on naturally occurring nucleic acids. Functionalized label is then conjugated to the target nucleic acid, either before or after it has been hybridized to the array of nucleic acids stably associated with the surface of a solid support. The subject methods find use in a variety of array based hybridization assays, including differential expression assays.
DEFINITIONS
The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. naturally occurring deoxyribonucleotides or ribonucleotides, as well as synthetic mimetics thereof which are also capable of participating in sequence specific, Watson-Crick type hybridization reactions, such as is found in peptide nucleic acids, etc.
The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
The term “target nucleic acid” means a nucleic acid of interest in a sample being tested with an array, where by “of interest” is meant that the presence or absence of target in the sample provides useful information, e.g. unique and defining characteristics, about the genetic profile of the cell(s) from which the sample is prepared. As such, target nucleic acids are not housekeeping genes or other types of genes which are present in a number of diverse cell types and therefore the presence or absence of which does not provide characterizing information about a particular cell's genetic profile.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and kits for labeling target nucleic acids for use in array based hybridization assays are provided. In the subject methods, a population of target nucleic acid is first generated from an initial nucleic acid source, e.g. mRNA, where each target nucleic acid in the population is characterized by having at least one reactive functionality that is not a moiety found on naturally occurring nucleic acids. Functionalized label is then conjugated to the target nucleic acid, either before or after it has been hybridized to array of nucleic acids stably associated with the surface of a solid support. The subject methods find use in a variety of array based hybridization assays, including differential expression assays. In further describing the invention, the subject methods are discussed first in greater detail followed by a review of the provided kits for use in practicing the subject methods.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein hav
Bochkariov Dmitry E.
Chenchik Alex
Bozicevic, Field & Francis
Clontech Laboratories, Inc.
Field Bret E.
Fredman Jeffrey
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