Isothermal methods for creating clonal single molecule arrays
First Claim
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1. A method for isothermally amplifying single stranded nucleic acid molecules immobilized on a planar solid surface comprising:
- i) providing a planar solid surface comprising at least one 5′
-end immobilized first single stranded nucleic acid template molecule comprising a sequence Y at the 5′
end and a sequence Z at the 3′
end and a plurality of first and second primers comprising sequences X and Y immobilized at their 5′
ends, wherein sequence X is hybridizable to sequence Z;
ii) annealing said at least one 5′
-end immobilized first single stranded nucleic acid template molecule to said first immobilized primers, wherein the first sequence Z of each template molecule is annealed to one of said first immobilized primers comprising sequence X;
iii) performing a primer extension reaction using primer annealed 5′
-end immobilized first single stranded nucleic acid template molecules to generate double stranded nucleic acid molecules comprising 5′
-end immobilized first and second single stranded nucleic acid molecules, wherein the 5′
-end immobilized second single stranded nucleic acid molecules are complementary copies of the 5′
-end immobilized first single stranded template nucleic acid molecules and each of the 5′
-end immobilized second single stranded nucleic acid molecules comprises a sequence at the 3′
end that is hybridizable to the second primer sequence Y;
iv) flowing a chemical denaturant across the planar solid surface to denature said double stranded nucleic acid molecules to generate 5′
-end immobilized first and second single stranded nucleic acid molecules;
v) removing the chemical denaturant and annealing said 5′
-end immobilized first and second single stranded nucleic acid molecules to said first and second immobilized primers comprising sequences X and Y;
vi) performing a primer extension reaction using primer annealed 5′
-end immobilized first and second single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules immobilized at both 5′
-ends; and
vii) repeating steps iv) through vi) to generate multiple copies of the nucleic acid molecules on said planar solid surface, wherein steps iv) through vi) are carried out at the same temperature.
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Abstract
The present invention is directed to a method for isothermal amplification of a plurality of different target nucleic acids, wherein the different target nucleic acids are amplified using universal primers and colonies produced thereby can be distinguished from each other. The method, therefore, generates distinct colonies of amplified nucleic acid sequences that can be analyzed by various means to yield information particular to each distinct colony.
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Citations
18 Claims
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1. A method for isothermally amplifying single stranded nucleic acid molecules immobilized on a planar solid surface comprising:
-
i) providing a planar solid surface comprising at least one 5′
-end immobilized first single stranded nucleic acid template molecule comprising a sequence Y at the 5′
end and a sequence Z at the 3′
end and a plurality of first and second primers comprising sequences X and Y immobilized at their 5′
ends, wherein sequence X is hybridizable to sequence Z;
ii) annealing said at least one 5′
-end immobilized first single stranded nucleic acid template molecule to said first immobilized primers, wherein the first sequence Z of each template molecule is annealed to one of said first immobilized primers comprising sequence X;
iii) performing a primer extension reaction using primer annealed 5′
-end immobilized first single stranded nucleic acid template molecules to generate double stranded nucleic acid molecules comprising 5′
-end immobilized first and second single stranded nucleic acid molecules, wherein the 5′
-end immobilized second single stranded nucleic acid molecules are complementary copies of the 5′
-end immobilized first single stranded template nucleic acid molecules and each of the 5′
-end immobilized second single stranded nucleic acid molecules comprises a sequence at the 3′
end that is hybridizable to the second primer sequence Y;
iv) flowing a chemical denaturant across the planar solid surface to denature said double stranded nucleic acid molecules to generate 5′
-end immobilized first and second single stranded nucleic acid molecules;
v) removing the chemical denaturant and annealing said 5′
-end immobilized first and second single stranded nucleic acid molecules to said first and second immobilized primers comprising sequences X and Y;
vi) performing a primer extension reaction using primer annealed 5′
-end immobilized first and second single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules immobilized at both 5′
-ends; and
vii) repeating steps iv) through vi) to generate multiple copies of the nucleic acid molecules on said planar solid surface, wherein steps iv) through vi) are carried out at the same temperature. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Current AssigneeSolexa Incorporated (Illumina Incorporated)
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Original AssigneeSolexa Incorporated (Illumina Incorporated)
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InventorsZhang, Lu, Rigatti, Roberto, Lloyd, David, Boutell, Jonathan, Barrost, Tobias, Schroth, Gary
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Application NumberUS11/725,597Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current506/016CPC Class CodesC12Q 1/6848 characterised by the means ...C12Q 2565/543 characterised by the use of...
