Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-10-11
2003-02-04
Zitomer, Stephanie W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S183000, C435S288300, C435S325000, C436S164000, C536S023500, C536S024310
Reexamination Certificate
active
06514693
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for detecting and optionally quantitating multiple copies of a repeat sequence in a nucleic acid molecule, preferably a telomere or centromere repeat sequence. The invention also relates to a method for determining the replicative potential of a cell.
BACKGROUND OF THE INVENTION
A large fraction of the DNA of all eukaryotes is made up of repeat sequences ranging from a few copies up to millions. Repeat functional sequences occur at the telomeres and centromeres of eukaryotic chromosomes. Telomeres and centromeres are important structural and functional elements of eukaryotic chromosomes. Telomeres are specialized nucleoprotein structures which are at the end of eukaryotic chromosomes (Blackburn, E H., Nature (London), 350:569-572, 1991). Telomeres in all vertebrates terminate in tandem arrays of the repeat sequence TTAGGG (Moyzis, R K., et al., Proc, Natl. Acad. Sci. USA 85:6622-6626, 1988). The repeat sequences are synthesized by the ribonucleoprotein enzyme telomerase, which is composed of both RNA and protein (Greider, C. W., & Blackburn, E. H. Cell 43:405-413, 1985; Morin, G B, Cell 59:521-529, 1989). In the absence of telomerase, telomeres shorten with cell divisions.
Telomeres have been shown to be critical for chromosome stability and function (Blackburn, E H., Nature (London), 350:569-572, 1991). Telomere loss has been shown to signal cell cycle arrest and chromosomal instability in yeast (Sandell, Ll, & Zakian, V A., Cell 57:633-643, 1989; and
Greider, C. W., & Blackburn, E. H. Cell 43:405-413, 1985). Telomeres in cells have been found to shorten with the age of the cell donor (Allsopp, R C. et al., Proc. Natl. Acad. Sci. USA 89, 10114-10118, 1992; Lindsey, J. et al., Mutat. Tes. 256:45-48, 1991, Vaziri, H., et al., Am. J. Hum. Genet. 52:661-667, 1993, Vaziri, H., et al., Proc. Natl. Acad. Sci. USA 91:9857-9861, 1994, and Hastie, N D., et al., Nature (London)., 346:866-868, 1990), Harley C B et al., Nature (London) 345:458-460, 1990; Harley, C B, Mutat. Res. 256:271-282, 1991), and this phenomenon has been implicated by some in aging (Harley, C B, Mutat. Res. 256:271-282, 1991), and in programmed cell death (Wright et al., Trends Genet 8:193-197, 1992). Abnormalities in telomeres have also been found in malignant cells (Meltzer et al., Nature Genetics 4:252-255, 1993).
The centromere region of mammalian chromosomes consists of tandem arrays of repetitive sequences which consist of various copy numbers of &agr; satellite (Willard, H F, Trends Genet. 3:192-198, 1987), &bgr; satellite (Waye, J S., & Willard H F., Proc. Natl. Acad. Sci. USA 86:6250-6254, 1989) and the three classic satellites I, II and III (Prosser J. et al., J. Mol. Biol. 187:145-155, 1989).
Fluorescent in situ hybridization (FISH) techniques have been used to obtain information about the presence of telomeric and centromeric repeat sequences in chromosome preparations. Meyne and Moyzis (in Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, ed. K. H. A. Choo, 1994, Humana Press Inc., Totowa, N.J. USA) used FISH with synthetic oligonucleotide probes to confirm the presence of repeat telomere and centromere sequences. Directly labeled oligonucleotides should be attractive probes for FISH because of their small size (good penetration properties), single strand nature (no renaturation of probe required), and reproducible, controlled synthesis. However, these probes are not widely used for FISH because of their limited hybridization efficiency. The hybridization efficiency of oligonucleotide probes is modest because of their small size, and because conditions required for hybridization of the probe typically also favour the renaturation of denatured target DNA sequences with the longer homologous complimentary strands, resulting in direct and unfavourable competition for the oligo probe. Oligomer probes also have limited use as probes for single copy sequences.
Significantly, the studies using FISH have not provided optimal visualization of telomeres in a chromosome preparation nor have they provided information about the length of telomere or centromere regions. Southern analysis of the terminal restriction fragment (TRF) length distribution has so far been the only tool for studying telomere length (Allshire R C et al., Nucl. Acid Res. 17:4611-4627 and Harley C B et al., Nature 345:458-60). However, Southern analysis has a number of technical limitations. The TRF's contain DNA other than the telomeric repeat sequences such as degenerate or non-TTAGGG as well as TTAGGG repeats in blocks other than at the distal end (Allshire et al., Nucl. Acid Res. 17:4611-4627; Counter et al., EMBO J. 11:1921-9, 1992 and Levy et al., J. Mol. Biol. 225:951-960, 1992). There is also interchromosomal variation in both non-TTAGGG and TTAGGG DNA in the TRF and variation in prior replicative histories in vitro or in vivo of cells in the population of cells required for TRF analysis, which makes it difficult to assess the relationship between cell senescence and TRF length in specific chromosome's and in specific cell types. This is a particular problem in cancer where a variable or unknown number of normal cells may be present in a tumor sample.
It is apparent from the above discussion that there is a need for reliable methods for in situ visualization and quantification of telomere and centromere regions in chromosome preparations.
SUMMARY OF THE INVENTION
The present inventor has developed a method for detecting repeat sequences in the genome of a single cell or in an individual chromosome. This has been illustrated by the detection of telomeric repeat sequences at the end of human chromosomes. It was significantly found that the illustrated method results in the highly sensitive and efficient staining of all telomeres in a chromosome preparation. The present inventor also found a high correlation between fluorescence intensity of telomere sister chromatids in metaphase chromosomes. The method thus allows for the highly sensitive and efficient detection of telomeres in a chromosome preparation, and/or it allows for the quantification of the length of telomeric arrays at individual ends. The method also permits one skilled in the art to determine the effect of telomere loss on cell viability and chromosome behaviour in a variety of disease states.
The method of the invention can also be used to detect and/or quantitate the length of other repeat sequences for example, centromere repeats and polymorphisms of the telomere and centromere repeats.
The method of the invention addresses many of the technical limitations of conventional in situ hybridization procedures. The limitations of using oligonucleotide probes in in situ hybridization has been overcome by using nucleic acid analogue probes, and conditions that allow hybridization of the probes but prevent the renaturation of target DNA. The method of the invention provides a more efficient and sensitive procedure than existing or conventional in situ hybridization methods using DNA or RNA oligo probes.
Therefore, broadly stated the present invention relates to a method for detecting multiple copies of a repeat sequence in a nucleic acid molecule comprising (a) treating the nucleic acid molecule with a probe which is a nucleic acid analogue which is capable of hybridizing to the repeat sequence in the nucleic acid molecule and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecule; and, (b) identifying probe hybridized to complementary repeat sequences in the nucleic acid molecule by directly or indirectly detecting the detectable substance.
In an embodiment of the invention, the detectable substance is a fluorophore, an image is formed of the probe hybridized to repeat sequences in the nucleic acid molecule, and, the multiple copies of the repeat sequences in the nucleic acid molecule are detected in the image.
The method of the invention may be used to quantitate the length of multiple copies
Gibbons Del Deo Dolan Griffinger & Vecchione
Tetramerics Biotechnology, Inc.
Zitomer Stephanie W.
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