Assays for nucleic acid sequences encoding telomerase
It might be expected that the groups that first cloned genes encoding telomerase have the dominant position(s) in this area. While other groups with patent positions in this area may still be able to dominate, Geron has the main published position effected by two U.S. patents (discussed below) and their related family counterparts. One of the patents US 6,808,880claims detecting nucleic acid sequences encoding at least a portion of telomerase by identifying a signature amino acid sequence. The motif is W-X12-FFY-X-TE, found in motif T, a motif unique to telomerases (Nakamura et al. 1997). Certainly the easier and more common methods to detect a nucleic acid are by hybridization or by amplification; both of these assay types are claimed in US 7,005,262.
The main claim directed to hybridization (claim 1) recites a method comprising:
- combining sample nucleic acids with a probe that anneals specifically to a nucleic acid sequence encoding hTERT (human telomerase) or a fragment of hTERT; and
- detecting hybridization.
The conditions for specific hybridization are detailed in the claim. The conditions (5-25°C below Tm in a 1M NaCl solution) aren’t very stringent. Most assuredly the probe will hybridize to related sequences. The limitation on the probe however is that the probe does not hybridize to the EST sequence derived from hTERT (SEQ ID No: 62) and originally found by scientists at Merck. Other independent claims (claims 22 and 27) are similar except that the probe hybridizes specifically to the EST sequence.
Claim 11 is broadly directed to an amplification (PCR) assay to detect sequences encoding human telomerase. In this claim:
- a sample is combined with a primer pair that amplifies nucleic acid encoding hTERT or a fragment of hTERT; and
- detecting amplified product.
Each of the primers contain at least 15 consecutive nucleotides (or complementary nucleotides) found in the sequence encoding hTERT as long as the primers are not derived from the EST sequence. In a related independent claim (claim 37) the primers are derived from the EST sequence. Other claims to amplification assays recite only “a polynucleotide primer” that either doesn’t hybridize (claim 10) or does hybridize (claim 32) to the EST sequence. In patent claim language, the indefinite article “a” means “one or more”. Thus, the claims reciting “a” primer are not limited to single primer amplification; instead, the amplification reaction can proceed using one, two, three, etc. primers. A plain reading of the claim however, limits one of the primers to not being derived from the EST sequence (claim 10) or to being derived from the EST sequence (claim 32), without limitation on the other primer(s).
Methods of detecting splice variants of human telomerase are claimed in US 6,916,642 owned by CAMBIA. In particular, claims 27 and 28 recite ways to determine a pattern of expression of the splice variants. The method:
- amplifies cDNA using primers that amplify a splice variant; and
- hybridizes the amplified product with a probe derived from one or more of the alternative listed “exon” sequences (claim 27) or derived from two or more of the specified sequences (claim 28).
|Patent Data||Title and relevant claims||Family Data|
University of Colorado
01 Oct 1996
19 Jan 2001
26 Oct 2004
01 Oct 2016
|Method for detecting polynucleotides encoding telomerase
Claim 1: A method for detecting the presence of polynucleotide sequences encoding at least a portion of telomerase in a biological sample, comprising the steps of: a) obtaining an amino acid sequence encoded in a polynucleotide contained in a biological sample; b) comparing the amino acid sequence with the telomerase amino acid motif W-X12 -FFY-X1 -TE, Wherein X is any amino acid; and then c) determining that the sample contains a polynucleotide encoding at least a portion of telomerase if the sequence obtained in step a) contains said telomerase amino acid motif.
|See Appendix 1|
01 Jul 1997
11 Feb 2000
12 Jul 2005
01 Jul 2017
|Vertebrate telomerase genes and proteins and uses thereof
Claim 28: A method of determining a pattern of telomerase RNA expression in cells, comprising,
(a) preparing cDNA from mRNA isolated from the cells,
(b) amplifying the cDNA using primers that amplify a splice variant of nucleic acid encoding human telomerase to form an amplified product and
(c) hybridizing the amplified product with two or more of the following:
all or at least 15 contiguous nucleotides of the sequence of region 1 (SEQ ID No: 23), all or at least 15 contiguous nucleotides of the sequence of region Î± (SEQ ID No: 25), all or at least 15 contiguous nucleotides of the sequence of region Î² (SEQ ID No: 27), all or at least 15 contiguous nucleotides of the sequence of region 2 (SEQ ID No: 29), all or at least 15 contiguous nucleotides of the sequence of region 3 (SEQ ID No: 30), all or at least 15 contiguous nucleotides of the sequence of region X (SEQ ID No: 32) or all or at least 15 contiguous nucleotides of the sequence of region Y (SEQ ID No: 18); or a complement thereof; and
(d) detecting hybridization;
therefrom determining the pattern of telomerase RNA expression.
AU 748442 B2
University of Colorado
18 Apr 1997
18 Jan 2002
28 Feb 2006
18 Apr 2017
|Methods for detecting nucleic acids encoding human telomerase reverse transcriptase
Claim 1: A method of identifying a nucleic acid that encodes human telomerase reverse transcriptase (hTRT) or fragment thereof in a sample, comprising:
a) combining the sample with a polynucleotide probe such that the probe hybridizes specifically to the nucleic acid if the nucleic acid encodes hTRT or fragment thereof:
b) detecting any hybrid formed as a result of a); and
c) identifying the nucleic acid as encoding hTRT or fragment thereof if the hybrid is detected;
wherein the probe hybridizes specifically to a DNA having the sequence of the hTRT encoding region of SEQ. ID NO:224 at 5° C. to 25° C. below Tm in aqueous solution at 1 M NaCl but does not hybridize to a DNA having the sequence of SEQ. ID NO:62 under the same reaction conditions;
wherein Tm is the melting temperature of double-stranded DNA having the sequence of said encoding region under the same reaction conditions.
Claim 11: A method of detecting a nucleic acid encoding hTRT or fragment thereof in a sample, comprising:
a) combining the sample with polynucleotide primers so as to prime amplification of nucleic acid encoding hTRT or fragment thereof if present in the sample;
b) detecting any amplified product formed as a result of a); and
c) identifying the nucleic acid as encoding hTRT or fragment thereof if the amplification product is detected:
wherein each of said primers consists essentially of a sequence identical or complementary to 15 or more consecutive nucleotides from the hTRT encoding region of SEQ. ID NO:224, but at least one of the primers does not consist sequence identical or complementary to 15 or more consecutive nucleotides from SEQ. ID NO:62.
|See Appendix 1|