Method for determination of telomere length
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Abstract
A method is described for determining telomere length of mammalian chromosomal DNA, which method comprises the steps: (a) annealing the 3′ end of a single-stranded oligonucleotide (hereinafter referred to as a ‘telorette’) to a single-stranded overhang of the telomere comprising the G-rich telomere strand (comprising TTAGGG repeat sequences) and covalently binding the telorette to the 5′ end of the C-rich telomeric strand (having CCCTAA repeat sequences) thereby forming a ligation product; (b) amplifying the ligation product formed in step (a) to form a primer extension product; and (c) detecting the length of the primer extension product(s) of step (b). Step (b) is preferably carried out by PCR using: (i) a first primer capable of annealing to a telomere-adjacent region of DNA but which first primer is not capable of annealing to the C-rich telomeric repeat sequence (CCCTAA); and (ii) a second primer (hereinafter referred to as a ‘teltail’ primer) identical to the 5′ end sequence of the telorette of step (a) which amplification is effected under conditions such that the first primer hybridises to the C-rich telomeric strand (comprising the CCCTAA repeats) and is extended to form a first primer extension product; and the teltail primer hybridises to the first primer extension product and is extended to form a second primer extension product. Also described are specific primers, including telorettes and teltails, for carrying out the method; a kit for use in the method; and the use of the method in determining telomere length and assessing biological conditions associated therewith.
10 Citations
58 Claims
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1-30. -30. (canceled).
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31. A method for determining telomere length of mammalian chromosomal DNA, which method comprises the steps of:
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(a) annealing a 3′
end of a single-stranded oligonucleotide linker or telorette to a single-stranded overhang of a telomere comprising a G-rich telomere strand having TTAGGG repeat sequences, and covalently binding the telorette to a 5′
end of a C-rich telomeric strand having CCCTAA repeat sequences thereby forming a ligation product;
(b) amplifying the ligation product to form at least one primer extension product; and
(c) detecting a length of the primer extension product. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57)
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54. A primer for use in determining telomere length of mammalian chromosomal DNA, comprising a 3′
- terminal having 6 to 12 nucleotide bases which are complementary to and capable of annealing to a G-rich telomeric overhang, and a 5′
terminal comprising 15 to 30 bases which are selected so as not to be complementary to the telomere but to be suitable for PCR amplification. - View Dependent Claims (55, 58)
- terminal having 6 to 12 nucleotide bases which are complementary to and capable of annealing to a G-rich telomeric overhang, and a 5′
Specification