Universal nucleotides for nucleic acid analysis
First Claim
Patent Images
1. A method of sequencing at least one target nucleic acid template comprising:
- (a) forming a reaction composition comprising at least one target nucleic acid template, at least one primer, at least one polymerase, at least one universal nucleotide, and at least one specific terminator comprising a label;
(b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides and one or more of the at least one specific terminators;
(c) separating one or more of the at least one primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and
(d) detecting one or more of the at least one primer extension products.
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Abstract
The invention includes methods and kits for making and analyzing primer extension products incorporating one or more universal bases, including methods and kits for nucleic acid sequencing and microsatellite analysis.
33 Citations
60 Claims
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1. A method of sequencing at least one target nucleic acid template comprising:
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(a) forming a reaction composition comprising at least one target nucleic acid template, at least one primer, at least one polymerase, at least one universal nucleotide, and at least one specific terminator comprising a label;
(b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides and one or more of the at least one specific terminators;
(c) separating one or more of the at least one primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and
(d) detecting one or more of the at least one primer extension products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 60)
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13. A method of sequencing at least one target nucleic acid template comprising:
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(a) forming at least one reaction composition comprising at least one target nucleic acid template, at least one primer comprising a label, at least one polymerase, at least one universal nucleotide, and at least one specific terminator;
(b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides and one or more of the at least one specific terminators;
(c) separating one or more of the at least one primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and
(d) detecting one or more of the at least one primer extension products. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A method of detecting a plurality of primer extension products comprising:
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(a) forming a reaction composition comprising at least one target nucleic acid template, at least one primer comprising a label, at least one polymerase, and at least one universal nucleotide;
(b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides;
(c) releasing one or more of the at least one primer extension products from one or more of the at least one target nucleic acid templates;
(d) repeating (b) and (c) to generate a plurality of primer extension products;
(e) separating one or more of the plurality of primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and
(f) detecting one or more of the plurality of primer extension products. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method of detecting a plurality of second primer extension products comprising:
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(a) forming a first reaction composition comprising at least one target nucleic acid template, at least one first primer, at least one specific nucleotide, and at least one first polymerase, wherein the first reaction composition does not include a universal nucleotide;
(b) incubating the reaction composition under appropriate conditions to generate at least one first primer extension product;
(c) releasing the one or more of at least one first primer extension products from one or more of the at least one target nucleic acid templates;
(d) repeating (b) and (c) to generate a plurality of first primer extension products;
(e) forming a second reaction composition comprising one or more of the plurality of first primer extension products, at least one second primer comprising a label, at least one second polymerase, and at least one universal nucleotide;
(f) incubating the reaction composition under appropriate conditions to generate at least one second primer extension product;
(g) releasing one or more of the at least one second primer extension products from one or more of the at least one first primer extension products;
(h) repeating (f) and (g) to generate a plurality of second primer extension products;
(i) separating one or more of the plurality of second primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and
(j) detecting one or more of the plurality of second primer extension products. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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- 48. A kit for sequencing a target nucleic acid template, comprising at least one universal nucleotide, at least one polymerase, and at least one specific terminator.
- 54. A kit for detecting a short tandem repeat in a target nucleic acid template, comprising at least one universal nucleotide, at least one polymerase, and at least one primer comprising a sequence that is complementary to a sequence adjacent to a short tandem repeat in the target nucleic acid template.
Specification