Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-20
2003-09-16
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310
Reexamination Certificate
active
06620586
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for analyzing nucleic acids. In particular, the invention provides for methods and combinations for analyzing nucleic acids in a plurality of samples using a plurality of detectably different signature labels and a probe that is hybridizable to each of the target nucleic acids. The invention also provides for methods for quantifying a nucleic acid by analyzing the amount of a photoactivatable label attached to the target nucleic acid.
BACKGROUND OF THE INVENTION
During the last decade there has been a revolution in the development of microarray technologies. By using a microarray chip, it is possible to get a genetic pattern, including gene expression profile and other information, which were unthinkable in the past. Unfortunately skilled personnel must carry out these methods and very expensive devices must be used to analyze such chips. One major deficiency of the array procedure and most other nucleic acid assay procedures used in a laboratory for identification of a target gene for diagnosis of diseases resides in the inability to analyze multiple samples or target genes by conducting a single hybridization with a probe or a probe cocktail without physically separating either the probes or the samples. This deficiency especially handicaps the utility of nucleic acid hybridization and array technology in high throughput assays by making the large number of assays more costly to perform. The currently available nucleic acid hybridization technologies use one sample one-hybridization format wherein only one sample can be used for hybridization with one probe or one array of nucleic acids immobilized on solid supports.
Accordingly, there is a need in the art for methods and compositions for analyzing nucleic acids wherein target nucleic acids in a plurality of samples can be analyzed concurrently or simultaneously in a single reaction step using a single probe or a cocktail of probes. The present invention addresses this and other related needs in the art.
BRIEF DESCRIPTION OF THE INVENTION
The present invention simplifies nucleic acid hybridization assays and eliminates the need of using complex array system for multi-sample and gene analysis and provides methods for a simplified hybridization analysis in a high throughput assay. The present invention uses, inter alia, signature compounds to label each sample. The labeled samples are then hybridized in a single reaction or container with a probe or a probe cocktail. After the hybridization step, analysis of the signature label in the hybrid duplex establishes the presence, absence and/or amount of the target nucleic acids and identifies which sample(s) contains the target nucleic acid(s).
In one aspect, the present invention is directed to a method for analyzing nucleic acids in a plurality of samples, which method comprises: a) attaching each of target nucleic acids, if there is one in a sample, in a plurality of samples with a signature label, preferably separately, whereby said target nucleic acid in each of said plurality of samples is attached to a detectably different signature label; b) pooling said labeled target nucleic acids in different samples into a single mixture; c) hybridizing each of said labeled target nucleic acids in said single mixture with a probe that is hybridizable to each of said labeled target nucleic acids in a single reaction to form a plurality of target nucleic acid/probe duplexes; and d) determining presence or absence, amount and/or identity of said target nucleic acid in each of said plurality of samples by analyzing presence or absence, amount and/or identity of said signature label in each of said target nucleic acid/probe duplexes.
In another aspect, the present invention is directed to a combination for analyzing nucleic acids in a plurality of samples, which combination comprises a plurality of detectably different signature labels, wherein each of said signature labels is capable of being attached to a target nucleic acid to be analyzed.
In still another aspect, the present invention is directed to a method for quantifying a nucleic acid, which method comprises attaching a label, and preferably a photoactivatable label, to a target nucleic acid and determining amount of said target nucleic acid by analyzing amount of said label attached to said target nucleic acid.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications and sequences from GenBank and other databases referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in applications, published applications and other publications and sequences from GenBank and other data bases that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
As used herein, “a” or “an” means “at least one” or “one or more.”
As used herein, “nucleic acid (s)” refers to deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) in any form, including inter alia, single-stranded, duplex, triplex, linear and circular forms. It also includes polynucleotides, oligonucleotides, chimeras of nucleic acids and analogues thereof. The nucleic acids described herein can be composed of the well-known deoxyribonucleotides and ribonucleotides composed of the bases adenosine, cytosine, guanine, thymidine, and uridine, or may be composed of analogues or derivatives of these bases. Additionally, various other oligonucleotide derivatives with nonconventional phosphodiester backbones are also included herein, such as phosphotriester, polynucleopeptides (PNA), methylphosphonate, phosphorothioate, polynucleotides primers and the like.
As used herein, “label” refers to any chemical group or moiety having a detectable physical property or any compound capable of causing a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes conversion of a substrate into a detectable product. The term “label” also encompasses compound that inhibit the expression of a particular physical property. The “label” may also be a compound that is a member of a binding pair, the other member of which bears a detectable physical property. Exemplary labels include mass groups, metals, fluorescent groups, luminescent groups, chemiluminescent groups, optical groups, charge groups, polar groups, colors, haptens, protein binding ligands, nucleotide sequences, radioactive groups, enzymes, particulate particles and a combination thereof.
As used herein, “detectably different signature label” means that the signature labels can be detected and distinguished from each other by any detection methods and/or instrumentation known in the art. Preferably, the signature labels can be detected and distinguished from each other in an environment suitable for nucleic acid hybridization. Also preferably, the signature labels can be quantified by any detection methods and/or instrumentation known in the art.
As used herein, “pooling said labeled target nucleic acids in different samples into a single mixture” means that target nucleic acid in each of a plurality of samples is mixed together in a single mixture, e.g., solution or fluid. The “pooling” step is conducted concurrently with or subsequent to the “labeling” step wherein the target nucleic acid in each of said plurality of samples is attached to a detectably different signature label. Preferably, “pooling” step is conducted subsequent to the “labeling” step. Although it is preferable that the target nucleic acids from all the samples to be analyzed can be “pooled” together into one single mixture before the hybridizing step, such “complete pooling” is not necessary. It is s
Applied Gene Technologies, Inc.
Benzion Gary
Hashemi Shar
Morrison & Foerster / LLP
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