Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-21
2003-03-18
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007200, C435S007210, C435S174000, C536S023100, C536S024300, C536S024310, C536S026600
Reexamination Certificate
active
06534266
ABSTRACT:
BACKGROUND OF THE INVENTION
Recent research on differential gene expression has compared overall gene expression in cancer cells with overall gene expression in normal counterpart cells (Zhang et al., 1998,
Science
276:1268-1272). Similarly, overall gene expression has been compared in cells undergoing different developmental programs (Chu et al., 1998,
Science
282:699-705). In such studies, it has been found that large numbers of genes are differentially expressed. For example, more than 500 transcripts are expressed at significantly different levels in cancer cells versus normal cells. In the case of cancer cells, it will be important to correlate the sequences identified as differentially expressed with actual events occurring at the cellular level or tissue level.
SUMMARY OF THE INVENTION
The invention provides an in situ hybridization method for detecting and specifically identifying transcription of a multiplicity of different target sequences in a cell. The method includes assigning a different bar code to at least five target sequences, with each target sequence containing at least one predetermined subsequence. Each bar code contains at least one fluorochrome, and at least one bar code comprises at least two different, spectrally distinguishable fluorochromes. A probe set specific for each target sequence is provided in the method. Each probe set contains a hybridization probe complementary to each subsequence in the target sequence. Each probe is labeled with a fluorochrome, and the fluorochromes in each probe set collectively correspond to the bar code for the target sequence of that probe set. The cell is contacted with a hybridization fluid containing a probe set specific for each target sequence. Following in situ hybridization, fluorochromes on the hybridized probe sets are detected, and spectrally distinguished. This provides separate detection of the transcription site of each target sequence being expressed. The fluorochromes present at each detected transcription site are related to a bar code, which identifies the target sequence at that transcription site.
Target sequences can include 3 or more, e.g., 4, 5, 6, or 7 predetermined, nonoverlapping subsequences. In some embodiments, at least one target sequence contains subsequences having lengths and spacing between each other so that the stoichiometry of fluorochromes on probes hybridized with the target sequence is determinable by quantitative fluorescence detection. This can be achieved, for example, by with each subsequence being 30 to 70 nucleotides long, and all the subsequences clustered within a 100-800 nucleotide segment of the target sequence. The region of clustering can be smaller, e.g., 200-600 nucleotides or 300-500 nucleotides. In some embodiments, each subsequence is about 50 nucleotides long, and all the subsequences are clustered within a 300-nucleotide segment of the target sequence. For maximization of total fluorescence intensity per transcription site, the 100-800 nucleotide segment is located in the 5′-most one third, or 5′-most quarter of the target sequence. Preferably, hybridization probes are labeled with fluorophores attached at intervals of about 5-10 nucleotides. Examples of fluorochromes useful in the invention are Cy2, fluorX, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein and Texas red. In some embodiments of the invention, a spectral imagining microscope is employed. Typically the cell is in interphase. The hybridization probe can be an oligonucleotide or a protein nucleic acid (PNA).
The invention also provides a probe set panel. The panel contains at least five probe sets, with each probe set being specific for a different target sequence, each of which contains at least one subsequence. Each probe set contains a hybridization probe complementary to each subsequence in the target sequence for which that probe set is specific. Each probe is labeled with a fluorochrome, so that the fluorochromes in each probe set collectively correspond to a bar code for the target sequence of that probe set.
As used herein, “bar code” means the predetermined, unique combination of fluorochromes assigned to a target sequence.
As used herein, “fluorochrome” means a particular fluorescent dye, e.g., Cy3, without regard to number of individual dye molecules, and without regard to chemical conjugation.
As used herein, “fluorophore” means an individual fluorescent dye molecule or conjugated moiety.
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. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, preferred methods and materials are described below. 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 detailed description and the claims.
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Albert Einstein College of Medicine of Yeshiva University
Fish & Richardson P.C.
Forman B J
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