Partially double-stranded oligonucleotide and method for forming oligonucleotide
First Claim
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1. A method of detecting a target nucleic acid, comprising:
- (a) preparing a single-stranded probe oligonucleotide immobilized on a carrier by sequentially;
(i) synthesizing a first and a second oligonucleotide capable of binding with each other through complementary sequences at the 3'"'"'-end regions of the first and the second oligonucleotides, and synthesizing a third and a fourth oligonucleotide, the third oligonucleotide being capable of binding with either the first or the second oligonucleotide through complementary sequences at the 5'"'"'-end regions of the first or the second oligonucleotide and the third oligonucleotide, and the fourth oligonucleotide being capable of binding with the third oligonucleotide through complementary sequences at the 3'"'"'-end regions of the third and the fourth oligonucleotides,(ii) binding the first, the second, the third and the fourth oligonucleotides to form a partially double-stranded oligonucleotide complex and then conducting an extension reaction in a reaction solution containing a nucleotide to which a fixing substance is bound, to polymerize nucleotides onto the 3'"'"'-end regions of the partially double-stranded oligonucleotide complex to form a double-stranded oligonucleotide in which a polymerized oligonucleotide portion of the double-stranded oligonucleotide is formed between the first and third oligonucleotide and between the second and fourth oligonucleotide, or between the first and fourth oligonucleotide and between the second and third oligonucleotide, in each of the polymerized oligonucleotide portions the fixing substance is incorporated,(iii) denaturing the double-stranded oligonucleotide to form a single-stranded probe oligonucleotide, and(iv) immobilizing the single-stranded probe oligonucleotide on the carrier by binding the fixing substance in each of the polymerized oligonucleotide portions to the carrier;
(b) hybridizing the single-stranded probe oligonucleotide immobilized on the carrier with a target single-stranded nucleic acid labeled with a labeling substance in a mixture of different nucleic acids, to obtain a double-stranded hybrid nucleic acid containing the labeling substance;
(c) separating the double-stranded hybrid nucleic acid from the mixture; and
(d) detecting the labeling substance in the separated double-stranded hybrid nucleic acid.
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Abstract
A method for forming an oligonucleotide comprises the steps of synthesizing a pair of oligonucleotides, one oligonucleotide having a base sequence portion comlementary to that of the other at a part of the terminal end region thereof; binding the pair of oligonucleotides at the complementary base sequence portions; and polymerizing nucleic acid bases onto the resulting pair of oligonuceotides. The method may further comprise the step of separating the oligonucleotides thus obtained into single strands.
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Citations
5 Claims
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1. A method of detecting a target nucleic acid, comprising:
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(a) preparing a single-stranded probe oligonucleotide immobilized on a carrier by sequentially; (i) synthesizing a first and a second oligonucleotide capable of binding with each other through complementary sequences at the 3'"'"'-end regions of the first and the second oligonucleotides, and synthesizing a third and a fourth oligonucleotide, the third oligonucleotide being capable of binding with either the first or the second oligonucleotide through complementary sequences at the 5'"'"'-end regions of the first or the second oligonucleotide and the third oligonucleotide, and the fourth oligonucleotide being capable of binding with the third oligonucleotide through complementary sequences at the 3'"'"'-end regions of the third and the fourth oligonucleotides, (ii) binding the first, the second, the third and the fourth oligonucleotides to form a partially double-stranded oligonucleotide complex and then conducting an extension reaction in a reaction solution containing a nucleotide to which a fixing substance is bound, to polymerize nucleotides onto the 3'"'"'-end regions of the partially double-stranded oligonucleotide complex to form a double-stranded oligonucleotide in which a polymerized oligonucleotide portion of the double-stranded oligonucleotide is formed between the first and third oligonucleotide and between the second and fourth oligonucleotide, or between the first and fourth oligonucleotide and between the second and third oligonucleotide, in each of the polymerized oligonucleotide portions the fixing substance is incorporated, (iii) denaturing the double-stranded oligonucleotide to form a single-stranded probe oligonucleotide, and (iv) immobilizing the single-stranded probe oligonucleotide on the carrier by binding the fixing substance in each of the polymerized oligonucleotide portions to the carrier; (b) hybridizing the single-stranded probe oligonucleotide immobilized on the carrier with a target single-stranded nucleic acid labeled with a labeling substance in a mixture of different nucleic acids, to obtain a double-stranded hybrid nucleic acid containing the labeling substance; (c) separating the double-stranded hybrid nucleic acid from the mixture; and (d) detecting the labeling substance in the separated double-stranded hybrid nucleic acid. - View Dependent Claims (2, 3)
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4. A method of separating a target nucleic acid from a mixture of different nucleic acids, comprising:
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(a) preparing a single-stranded probe oligonucleotide immobilized on a carrier by sequentially; (i) synthesizing a first and a second oligonucleotide capable of binding with each other through complementary sequences at the 3'"'"'-end regions of the first and the second oligonucleotides, and synthesizing a third and a fourth oligonucleotide, the third oligonucleotide being capable of binding with either the first or the second oligonucleotide through complementary sequences at the 5'"'"'-end regions of the first or the second oligonucleotide and the third oligonucleotide, and the fourth oligonucleotide being capable of binding with the third oligonucleotide through complementary sequences at the 3'"'"'-end regions of the third and the fourth oligonucleotides, (ii) binding the first, the second, the third and the fourth oligonucleotides to form a partially double-stranded oligonucleotide complex and then conducting an extension reaction in a reaction solution containing a nucleotide to which a fixing substance is bound, to polymerize nucleotides onto the 3'"'"'-end regions of the partially double-stranded oligonucleotide complex to form a double-stranded oligonucleotide in which a polymerized oligonucleotide portion of the double-stranded oligonucleotide is formed between the first and third oligonucleotide and between the second and fourth oligonucleotide, or between the first and fourth oligonucleotide and between the second and third oligonucleotide, in each of the polymerized oligonucleotide portions the fixing substance is incorporated, (iii) denaturing the double-stranded oligonucleotide to form a single-stranded probe oligonucleotide, and (iv) immobilizing the single-stranded probe oligonucleotide on the carrier by binding the fixing substance in each of the polymerized oligonucleotide portions to the carrier; (b) hybridizing the single-stranded probe oligonucleotide immobilized on the carrier with a target single-stranded nucleic acid in the mixture of different nucleic acids, to obtain a double-stranded hybrid nucleic acid; and (c) separating the double-stranded hybrid nucleic acid from the mixture. - View Dependent Claims (5)
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Specification
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Current AssigneeCanon Kabushiki Kaisha (Canon Inc.)
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Original AssigneeCanon Kabushiki Kaisha (Canon Inc.)
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InventorsIwashita, Harumi, Kato, Kinya, Yamamoto, Nobuko, Sakuranaga, Masanori
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Primary Examiner(s)Elliott, George C.
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Assistant Examiner(s)MCKELVEY, TERRY ALAN
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Application NumberUS07/974,303Time in Patent Office2,184 DaysField of Search435/6, 435/91.2, 536/25.4US Class Current435/6.18CPC Class CodesC12N 15/10 Processes for the isolation...
