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US 5,856,096 AGranted
Rapid and sensitive assays for detecting and distinguishing between processive and non-processive telomerase activities
AssigneeCTRC Research Foundation
InventorsBradford E. Windle, Ming Qiu, Shih-Fong Chen, Terace M. Fletcher, Ira Maine
Life of this patent
Estimating the term…
Abstract
Improved telomerase activity assays are provided in which a ligation sequential reaction (LSR) or BrdUTP are used to identify a telomerase specific product. These assays are useful in diagnosing various cancers and determining the clinical prospects for cancer patients. In addition, the assays can be used to screen for substances that interfere with telomerase activity.
Claims42
A method for detecting telomerase activity in a sample comprising the steps of:
- (i) obtaining said sample;
- (ii) contacting said sample with a telomerase primer and dNTPs under conditions permitting formation of a telomerase product;
- (iii) contacting the telomerase product of step (ii) with a first oligonucleotide and a second oligonucleotide under conditions permitting hybridization, wherein said first and said second oligonucleotides hybridize to said telomerase product such that no single-stranded region intervenes between said first and said second oligonucleotides to form a hybridized product;
- (iv) contacting the hybridized product and oligonucleotides with a ligase;
- and
- (v) detecting the ligated form of said first and said second oligonucleotides.
3 more elements
A method for detecting telomerase activity in a sample comprising the steps of:
- (i) obtaining said sample;
- (ii) contacting said sample with a telomerase primer and dNTPs, wherein said dNTPs include BrdUTP or hapten-labeled nucleotides;
- and
- (iii) determining the incorporation of BrdU or hapten-labeled nucleotides into a telomerase product of step (ii).
1 more element
A method for determining telomere length comprising the steps of:
- (i) providing a genomic DNA;
- (ii) contacting said genomic DNA with a plurality of oligonucleotides, under conditions permitting hybridization thereof, wherein said oligonucleotides hybridize to telomeric DNA such that no single-stranded regions intervenes between said hybridized oligonucleotides;
- (iii) contacting the hybridized telomeric DNA and oligonucleotides with a ligase;
- and
- (iv) determining the length of the ligated form of said oligonucleotides.
2 more elements
A method for obtaining a substance having telomerase-inhibiting activity comprising the steps of:
- (i) providing a telomerase preparation;
- (ii) contacting said telomerase preparation with a telomerase primer and dNTPs in the presence of said substance to form a telomerase product;
- (iii) contacting said telomerase product of step (ii) with a first oligonucleotide and a second oligonucleotide under conditions permitting hybridization, wherein said first and said second oligonucleotides hybridize to said telomerase product such that no single-stranded region intervenes between said first and said second oligonucleotides to form a hybridized product;
- (iv) contacting the hybridized product and oligonucleotides with a ligase;
- (v) detecting the ligated form of said first and said second oligonucleotides;
- (vi) comparing the amount of telomerase product of step (iii) with the amount of telomerase product synthesized in the absence of said substance;
- and
- (vii) obtaining said substance.
5 more elements
A method for obtaining a substance having telomerase-inhibiting activity comprising the steps of:
- (i) providing a telomerase preparation;
- (ii) contacting said sample with a telomerase primer and dNTPs, wherein said dNTPs include BrdU or hapten-labeled nucleotides and one or more candidate inhibitor substances to form a telomerase product;
- (iii) determining the incorporation of BrdU or hapten-labeled nucleotides into the telomerase product of step (ii);
- (iv) comparing the amount of telomerase product of step (ii) with the amount of telomerase product synthesized in the absence of each of said candidate inhibitor substances;
- and
- (v) obtaining said candidate inhibitor substance or substances.
3 more elements
A method for diagnosing cancer in a subject comprising the steps of:
- (i) obtaining a tissue sample from said subject;
- (ii) contacting said tissue sample with a telomerase primer and dNTPs to form a mixture;
- (iii) contacting the mixture of part (ii) with a first oligonucleotide and a second oligonucleotide under conditions permitting hybridization, wherein said first and said second oligonucleotides hybridize to a telomerase product such that no single-stranded region intervenes between said first and said second oligonucleotides;
- (iv) contacting the mixture of part (iii) with a ligase;
- and
- (v) determining if a ligated form of said first and said second oligonucleotides is present,
- whereby the presence of a ligated form of said first and said second oligonucleotides is indicative of cancer.
4 more elements
A method for determining the processivity of a telomerase comprising the steps of:
- (i) obtaining a sample;
- (ii) contacting separate portions of said sample with
- (a) a single repeat telomerase primer and dNTPs' and
- (b) a two-repeat telomerase primer and dNTP's;
- (iii) determining the presence or absence of telomerase extension products in each of said portions,
- whereby the presence of telomerase extension products in both the portions indicates processive telomerase activity, and whereby the presence of telomerase extension products in only the two-repeat primed portion indicates non-processive telomerase activity.
3 more elements
A method for determining processivity of a telomerase comprising the steps of:
- (i) obtaining a sample;
- (ii) contacting separate portions of said sample with
- (a) a telomerase primer not ending in TTAG and dNTPs;
- and
- (b) a telomerase primer ending in TTAG and dNTPs;
- and
- (iii) determining the presence or absence of telomerase extensions products in said portions,
- whereby the presence of telomerase extension products in the portion comprising the TTAG telomerase primer indicates processive telomerase activity, and whereby the presence of telomerase extension products in the portion comprising the telomerase primer not ending in TTAG indicates total telomerase activity.
5 more elements
Dependencies are inferred from the claim text.