Highly sensitive multimeric nucleic acid probes
First Claim
1. A detectably labeled oligonucleotide multimer comprising a 5'"'"' nonrepeated region comprising a nucleotide sequence that binds a first target molecule in a sample, followed by multiple contiguous copies of a repeated oligonucleotide, wherein at least one copy of the repeated oligonucleotide comprises at least one copy of a detectable label.
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Abstract
The present invention provides detectably labeled RNA and DNA oligonucleotide multimers useful as diagnostic probes in medical, biological and chemical applications. A method for synthesizing DNA and RNA oligonucleotides, oligonucleotide multimers, and analogs, preferably those that are detectably labeled, is also provided. Oligonucleotide synthesis is performed by combining a circular single-stranded oligonucleotide template with an effective polymerase and at least two types of nucleotide triphosphate, without the addition of auxiliary proteins, to yield an oligonucleotide multimer comprising multiple copies of a repeated oligonucleotide sequence.
156 Citations
66 Claims
- 1. A detectably labeled oligonucleotide multimer comprising a 5'"'"' nonrepeated region comprising a nucleotide sequence that binds a first target molecule in a sample, followed by multiple contiguous copies of a repeated oligonucleotide, wherein at least one copy of the repeated oligonucleotide comprises at least one copy of a detectable label.
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34. A method for synthesizing a detectable labeled oligonucleotide multimer comprising:
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(a) providing a single-stranded circular template comprising at least one copy of a nucleotide sequence complementary to the nucleotide sequence of a selected oligonucleotide; (b) annealing an effective amount of an oligonucleotide primer to the single-stranded circular template to yield a primed single-stranded circular template; and (c) combining the primed single-stranded circular template with an effective amount of at least two types of nucleotide triphosphate, at least one of the at least two types of nucleotide triphosphate comprising a detectable label and an effective amount of a polymerase enzyme, without the addition of auxiliary proteins, to yield a detectably labeled oligonucleotide multimer comprising a 5'"'"' nonrepeated region comprising a nucleotide sequence that binds a first target molecule followed by multiple contiguous copies of the selected oligonucleotide, wherein at least one copy of the selected oligonucleotide comprises at least one copy of a detectable label. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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- 60. A detectably labeled oligonucleotide multimer comprising multiple copies of a fluorescent label, the oligonucleotide multimer further comprising a 5'"'"' nonrepeated region comprising a nucleotide sequence that binds a target molecule in a sample, followed by multiple contiguous copies of a repeated oligonucleotide wherein at least one copy of the repeated oligonucleotide comprises at least one copy of a fluorescent label and wherein the multiple copies of the fluorescent label are positioned within the oligonucleotide multimer such that each copy of the fluorescent label is separated from every other copy of the fluorescent label on the oligonucleotide multimer by a separation distance of at least about 10 nucleotides.
Specification