Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-04-24
2001-07-31
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S024300, C536S025320, C536S026600
Reexamination Certificate
active
06268184
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention pertains to the field to the field of cytogenetics. More particularly this invention pertains to the identification of a amplification at about 20q13 that is a good prognostic indicator of various cancers. In addition, this invention provides a number of probes specific for the 20q13 amplicon.
Chromosome abnormalities are often associated with genetic disorders, degenerative diseases, and cancer. In particular, the deletion or multiplication of copies of whole chromosomes or chromosomal segments, and higher level amplifications of specific regions of the genome are common occurrences in cancer. See, for example Smith, et al.,
Breast Cancer Res. Treat
., 18: Suppl. 1: 5-14 (1991, van de Vijer & Nusse,
Biochim. Biophys. Acta
. 1072: 33-50 (1991), Sato, et al.,
Cancer. Res
., 50: 7184-7189 (1990). In fact, the amplification and deletion of DNA sequences containing proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumorigenesis. Dutrillaux, et al.,
Cancer Genet. Cytogenet
., 49: 203-217 (1990). Clearly the identification of amplified and deleted regions and the cloning of the genes involved is crucial both to the study of tumorigenesis and to the development of cancer diagnostics.
The detection of amplified or deleted chromosomal regions has traditionally been done by cytogenetics. Because of the complex packing of DNA into the chromosomes, resolution of cytogenetic techniques has been limited to regions larger than about 10 Mb; approximately the width of a band in Giemsa-stained chromosomes. In complex karyotypes with multiple translocations and other genetic changes, traditional cytogenetic analysis is of little utility because karyotype information is lacking or cannot be interpreted. Teyssier, J. R.,
Cancer Genet. Cytogenet
., 37: 103 (1989). Furthermore conventional cytogenetic banding analysis is time consuming, labor intensive, and frequently difficult or impossible.
More recently, cloned probes have been used to assess the amount of a given DNA sequence in a chromosome by Southern blotting. This method is effective even if the genome is heavily rearranged so as to eliminate useful karyotype information. However, Southern blotting only gives a rough estimate of the copy number of a DNA sequence, and does not give any information about the localization of that sequence within the chromosome.
Comparative genomic hybridization (CGH) is a more recent approach to identify the presence and localization of amplified/deleted sequences. See Kallioniemi, et al.,
Science
, 258: 818 (1992). CGH, like Southern blotting, reveals amplifications and deletions irrespective of genome rearrangement. Additionally, CGH provides a more quantitative estimate of copy number than Souther blotting, and moreover also provides information of the localization of the amplified or deleted sequence in the normal chromosome.
Using CGH, the chromosomal 20q13 region has been identified as a region that is frequently amplified in cancers (see, e.g. copending application U.S. Ser. No. 08/132,808, filed on Oct. 6, 1993). Initial analysis of this region in breast cancer cell lines identified a region approximately 2 Mb on chromosome 20 that is consistently amplified.
SUMMARY OF THE INVENTION
The present invention relates to the identification of a narrow region (about 600 kb) within a 2 Mb amplicon located at about chromosome 20q13 (more precisely at 20q13.2)that is consistently amplified in primary tumors. In addition this invention provides a contig (a series of clones that contiguously spans this amplicon) as well as sequence information for a large number of exons and cDNAs located within the contig. The contig or components thereof can be used to prepare probes specific for the amplicon. The probes can be used to detect chromosomal abnormalities at 20q13.
Thus, in one embodiment, this invention provides a method of detecting a chromosome abnormality (e.g., an amplification or a deletion) at about position FLpter 0.825 on human chromosome 20 (20q13.2). The method involves contacting a chromosome sample from a patient with a composition consisting essentially of one or more labeled nucleic acid probes each of which binds selectively to a target polynucleotide sequence at about position FLpter 0.825 on human chromosome 20 under conditions in which the probe forms a stable hybridization complex with the target sequence; and detecting the hybridization complex. The step of detecting the hybridization complex can involve determining the copy number of the target sequence. The probe preferably comprises a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected from the nucleic acids listed in Table 1 or Table 2. Even more preferably, the probe is one or more nucleic acids selected from the nucleic acids listed in Table 1 or Table 2. The probe is preferably labeled, and is more preferably labeled with digoxigenin or biotin. In one embodiment, the hybridization complex is detected in interphase nuclei in the sample. Detection is preferably carried out by detecting a fluorescent label (e.g., FITC, fluorescein, or Texas Red). The method can further involve contacting the sample with a reference probe which binds selectively to a chromosome 20 centromere.
In another embodiment, this invention provides for probes that specifically bind to the 20q13 amplicon. Thus, this invention provides for a composition comprising a labeled nucleic acid probe which binds selectively to a target polynucleotide sequence at about FLpter 0.825 on human chromosome 20. The probes comprise one or more nucleic acids selected from the group consisting of the nucleic acids listed in Table 1 or Table 2. In a preferred embodiment, the probes are labelled with digoxigenin or biotin.
This invention also provides for kits for the detection of a chromosomal abnormality at about position FLpter 0.825 on human chromosome 20. The kits include a compartment which contains a labeled nucleic acid probe which binds selectively to a target polynucleotide sequence at about FLpter 0.825 on human chromosome 20. The probe preferably includes at least one nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected the nucleic acids listed in Table 1 or Table 2. Even more preferably, the probes comprise one or more nucleic acids selected from the nucleic acids listed in Table 1 or Table 2. In a preferred embodiment, the probes are labelled with digoxigenin or biotin. The kit may further include a reference probe specific to a sequence in the centromere of chromosome 20.
Definitions
A “chromosome sample” as used herein refers to a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques.
“Nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
“Subsequence” refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
A “probe” or a “nucleic acid probe”, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a target can be detected. The probe is labeled as described below so that its binding to the target can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, for example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the 20q13 amplicon as described herein. The probe may be processed in some manner, for example, by blocking or rem
Collins Colin
Gray Joe W.
Kallioniemi Ol'li-Pekka
Pinkel Daniel
Tanner Minna M.
Hunter Tom
Jones W. Gary
The Law Office of Jonathan Alan Quine
The Regents of the University of California
Tung Joyce
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