Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-25
2003-04-08
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S025320
Reexamination Certificate
active
06544738
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of nucleic acid detection and specifically to a method for identifying a specific nucleic acid in a population of nucleic acids using a cleavable molecular tag linked to an oligonucleotide.
BACKGROUND OF THE INVENTION
An important part of functional genomics is the analysis of gene transcription in a large population of nucleic acids. Transcription analysis can potentially allow for the determination the identity of each gene expressed in a cell and the relative amount of transcript expressed compared with a control sample. Such analyses can thus reveal the “gene state” of a cell type or organism. However, quantitative analyses of transcription levels in a cell can be limited by the relatively large amount of the input RNA sample material needed, and by the wide variation in the abundance of different transcripts in a population of nucleic acid molecules, e.g., in the relative abundance of RNA molecules present in a single cell. The relative abundance, or dynamic range, can vary about four logs (10,000-fold) in RNA from populations of homogeneous cells or single cell assays. The range may vary for an additional three orders or more of magnitude for heterogenous cell samples.
There exists a need for a method detecting specific nucleic acid sequences in small amounts of a population of nucleic acid sequence, and whose abundance can vary over several orders of magnitude.
SUMMARY OF THE INVENTION
The invention is based on the discovery that cleavable tags attached to oligonucleotides can be used to identify specific nucleic acids in a population or collection of nucleic acids. In the methods described herein, tagged oligonucleotides are hybridized with a population or collection of nucleic acids. Hybridized oligonucleotides are then separated from non-hybridized oligonucleotides, and the tag is cleaved from the hybridized oligonucleotide and its identity determined. The identity of the tag thus reveals the identity of the oligonucleotide and the nucleic acid in the population or collection of nucleic acids to which the oligonucleotide hybridized.
The invention provides methods of detecting a specific nucleic acid in a population or collection of nucleic acids. In other embodiments, the invention provides compositions of tagged oligonucleotides and kits comprising tagged oligonucleotides for detecting specific nucleic acid sequences.
Among the advantages of the invention is increased sensitivity in detecting specific nucleic acid sequences in small amounts of an input population or collection of nucleic acid sequences. Another advantage of the invention is the ability to detect nucleic acids whose relative abundance may differ over several orders of magnitude in a nucleic acid sample.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following description, and from the claims.
REFERENCES:
patent: 5367066 (1994-11-01), Urdea et al.
patent: 5639603 (1997-06-01), Dower et al.
patent: 5770367 (1998-06-01), Southern et al.
patent: 6027890 (2000-02-01), Ness et al.
patent: 95/04160 (1995-02-01), None
Benzion Gary
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Tung Joyce
Vacchiano Emanuel J.
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