Method for detecting multiple copies of a repeat sequence in a nucleic acid molecule

Abstract

A method for detecting or quantitating multiple copies of a repeat sequence in a nucleic acid molecule involving 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. The nucleic acid molecule is treated with the probe under conditions permitting the probe to hybridize to the repeat sequences in the nucleic acid molecule. Probe hybridized to complementary repeat sequences is identified in the nucleic acid molecule by directly or indirectly detecting the detectable substance. The method is preferably used for quantitating multiple copies of a repeat sequence in a nucleic acid molecule, preferably a telomere or centromere repeat sequence. Novel probes for use in the method of the invention and kits are described.

I claim:  
     
         1 . 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 repeat sequences 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 repeat sequences in the nucleic acid molecule by directly or indirectly detecting the detectable substance.  
     
     
         2 . A method as claimed in  claim 1 , wherein the detectable substance is a fluorophore, isotope, or chemiluminescent compound.  
     
     
         3 . A method as claimed in  claim 1 , wherein the detectable substance is a fluorophore, and in (b) probe hybridized to repeat sequences is determined by forming an image of the probe hybridized to repeat sequences in the nucleic acid molecule, and, detecting the multiple copies of the repeat sequences in the nucleic acid molecule by detecting fluorescence in the image.  
     
     
         4 . A method as claimed in  claim 1 , wherein the detectable substance is an enzyme or hapten.  
     
     
         5 . A method for quantitating the length of 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 repeat sequences 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; (b) identifying probe hybridized to repeat sequences in the nucleic acid molecule by detecting a signal produced directly or indirectly by the detectable substance; and, (c) quantitating the length of the multiple copies of the repeat sequences in the nucleic acid molecule based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance and the length of the multiple copies of the repeat sequence.  
     
     
         6 . A method as claimed in  claim 1  or  5  wherein, the repeat sequence is a telomere or centromere repeat sequence, most preferably a human telomeric repeat sequence.  
     
     
         7 . A method as claimed in  claim 1  or  3  wherein, the repeat sequence is TTAGGG and the probe contains the sequence CCCTAA.  
     
     
         8 . A method for quantitating the length of multiple copies of the telomere repeat sequence TTAGGG in a nucleic acid molecule comprising (a) treating the nucleic acid molecule with a probe which is a nucleic acid analogue comprising the sequence CCCTAA which is labelled with a fluorophore, in the presence of a blocking reagent and a denaturing agent, preferably formamide, most preferably 70% formamide, and permitting the probe to hybridize to TTAGGG telomere repeat sequences in the nucleic acid molecule, and, (b) forming an image of probe hybridized to TTAGGG telomere repeat sequences in the nucleic acid molecule; and, (c) quantitating the length of the TTAGGG telomere repeat sequences in the nucleic acid molecule based on the direct relationship between fluorescence intensity and the length of the multiple copies of the telomere sequence.  
     
     
         9 . A method for determining the replicative potential of a cell by quantitating the length of multiple copies of a telomere repeat sequence TTAGGG in nucleic acid molecules in the cell comprising (a) treating the nucleic acid molecules with a probe which is a nucleic acid analogue comprising the sequence CCCTAA and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to TTAGGG telomere repeat sequences in the nucleic acid molecules; (b) identifying probe hybridized to TTAGGG telomere repeat sequences in the nucleic acid molecule by detecting a signal produced directly or indirectly by the detectable substance; (c) quantitating the length of the multiple copies of the TTAGGG telomere repeat sequence based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance and the length of the multiple copies of the telomere repeat sequence and (d) determining the replicative potential by comparing the quantitated length of the multiple copies of the telomere repeat sequence with the length of multiple copies of the telomere repeat sequence associated with cells having a known replicative potential.  
     
     
         10 . A method for determining the effect of a substance on telomerase activity comprising (a) treating cells having telomerase activity with a probe which is a nucleic acid analogue comprising the sequence CCCTAA and which is labelled with a detectable substance, preferably a fluorophore, and with a substance suspected of affecting telomerase activity, under conditions permitting the probe to hybridize to TTAGGG telomere repeat sequences in nucleic acid molecules in the cells; (b) identifying probe hybridized to TTAGGG telomere repeat sequences in the nucleic acid molecules by detecting a signal produced directly or indirectly by the detectable substance; (c) quantitating the length of multiple copies of the TTAGGG telomere repeat sequence in the nucleic acid molecules based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance and the length of the multiple copies of the telomere repeat sequence, and (d) determining the effect of the substance by comparing the quantitated length of the multiple copies of the telomere repeat sequence with the length of multiple copies of the telomere repeat sequence quantitated for the preparation in the absence of the substance.  
     
     
         11 . A method for quantitating multiple copies of repeat sequences in nucleic acid molecules in cells in suspension comprising; (a) treating the cells 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; (b) subjecting the treated cells to flow cytometry or image cytometry to detect the detectable substance and produce a signal corresponding to the amount of probe hybridized to repeat sequences in the nucleic acid molecule; and (c) quantitating the length of the multiple copies of the repeat sequences in the nucleic acid molecule based on the relationship between the intensity of the signal and the length of the multiple copies of the repeat sequence.  
     
     
         12 . A method as claimed in  claim 11  wherein the repeat sequence is a telomere repeat sequence.  
     
     
         13 . A method as claimed in  claim 12  wherein the telomere repeat sequence is TTAGGG and the probe contains the sequence CCCTAA.  
     
     
         14 . A method as claimed in  claim 11 , wherein the detectable substance is a fluorophore.  
     
     
         15 . A method as claimed in  claim 11  wherein the cells are lymphocytes.  
     
     
         16 . A method for distinguishing normal cells from a tumor cells in a cell suspension containing normal cells and tumor cells comprising: (a) treating the cells in the cell suspension with a probe which is a nucleic acid analogue which is capable of hybridizing to a repeat sequence in nucleic acid molecules in the nuclei of the cells and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecule; (b) subjecting the treated cells to flow cytometry or image cytometry to detect the detectable substance and produce a signal corresponding to the amount of probe hybridized to repeat sequences in the nucleic acid molecule; and (c) determining whether the cells are normal cells or tumor cells by comparing the signal with a signal obtained for known normal cells or tumor cells.  
     
     
         17 . A Peptide Nucleic Acid (PNA) probe comprising the following sequence: TTAGGG, CCCTAA, CCCCAA, CCCCAAAA, CCCACA, CCCTAAA, CCCCT, or CCATT.  
     
     
         18 . A Peptide Nucleic Acid (PNA) probe comprising the following sequence:CCCTAACCCTAA, CCCTAACCCTAACCCTAA, TTAGGGTTAGGG, or TTAGGGTTAGGGTTAGGG.  
     
     
         19 . A kit comprising at least one 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, and reagents, buffers and instructions for performing the method as claimed in  claim 1.