Dual-labeled nucleotides
First Claim
1. A nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to a phosphate moiety that is cleaved off when said nucleotide is enzymatically incorporated into a polynucleotide.
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Abstract
The present invention relates to a dual-labeled nucleotide comprising a fluorescent label and a quencher of that fluorescent label, wherein the quencher is attached to a phosphate moiety that is cleaved off when the nucleotide is enzymatically incorporated into a polynucleotide. The invention further relates to methods of labeling a polynucleotide molecule, as well as methods for identifying one or more residues of a polynucleotide using the dual-labeled nucleotide analogs of the present invention and kits comprising dual-labeled nucleotide analogs according to the invention.
149 Citations
57 Claims
- 1. A nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to a phosphate moiety that is cleaved off when said nucleotide is enzymatically incorporated into a polynucleotide.
- 13. A nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to the polyphosphate moiety of said nucleotide and said fluorescent label is attached to the nucleobase of said nucleotide.
- 22. A dual-labeled nucleotide analog having a general structure selected from the group consisting of
- 28. A method of synthesizing a polynucleotide, the method comprising contacting a nucleic acid polymerase enzyme with a nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to a phosphate moiety that is cleaved off when said nucleotide is enzymatically incorporated into a polynucleotide, under conditions permitting the extension of a nucleic acid primer annealed to a template nucleic acid, wherein said nucleotide is thereby incorporated into said nucleic acid primer.
- 37. A method of labeling a polynucleotide, the method comprising contacting a nucleic acid polymerase enzyme with a nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to a phosphate moiety that is cleaved off when said nucleotide is enzymatically incorporated into a polynucleotide, under conditions permitting the extension of a nucleic acid primer annealed to a template nucleic acid, whereby said dual-labeled nucleotide analog is incorporated into said nucleic acid primer, thereby labeling said polynucleotide.
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44. A method of determining sequence information about a template polynucleotide, the method comprising
(a) annealing an oligonuclotide primer to a template polynucleotide to generate an annealed primer; -
(b) contacting said annealed primer and said template of step (a) with a nucleic acid polymerase enzyme in the presence of a nucleotide comprising a fluorescent label and a quencher of said fluorescent label, wherein said quencher is attached to a phosphate moiety that is cleaved off when said nucleotide is enzymatically incorporated into a polynucleotide, under conditions sufficient to permit the extension of said primer by said nucleic acid polymerase enzyme; and
(c) detecting the incorporation of said dual-labeled nucleotide analog into said primer, wherein said incorporation determines sequence information about said template polynucleotide. - View Dependent Claims (45, 46, 47, 48, 49, 50, 51, 52, 53)
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Specification