Isothermal amplification of nucleic acids on a solid support
First Claim
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1. A method for isothermally amplifying single stranded nucleic acid molecules immobilized on a solid support comprising:
- i) providing a plurality of different nucleic acid template molecules and attaching a sequence Y to a 5′
end of each of the nucleic acid template molecules and a sequence Z to a 3′
end of each of the nucleic acid template molecules to generate different nucleic acid template molecules comprising the same sequence Y at the 5′
end and the same sequence Z at the 3′
end;
ii) providing a solid surface comprising a plurality of first primers comprising sequence X immobilized at their 5′
ends and a plurality of second primers comprising sequence Y immobilized at their 5′
ends, wherein sequence X is hybridizable to sequence Z;
iii) contacting the nucleic acid template molecules comprising sequence Y at the 5′
end and sequence Z at the 3′
end with the solid surface to generate at least one 5′
-end immobilized first single stranded nucleic acid template molecule comprising the sequence Y at the 5′
end and the sequence Z at the 3′
end;
iv) annealing said at least one 5′
-end immobilized first single stranded nucleic acid template molecule to said first immobilized primers, wherein sequence Z of each template molecule is annealed to one of said first immobilized primers comprising sequence X;
v) performing a primer extension reaction using said first immobilized primers that are annealed to said 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;
vi) denaturing said double stranded nucleic acid molecules to generate 5′
-end immobilized first and second single stranded nucleic acid molecules;
vii) annealing said 5′
-end immobilized first and second single stranded nucleic acid molecules to said first immobilized primers comprising sequence X and said second immobilized primers comprising sequence Y, respectively;
viii) 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;
ix) repeating steps vi through viii to generate DNA colonies, wherein each DNA colony comprises multiple copies of the 5′
-end immobilized first and second single stranded nucleic acid molecules on said solid surface, wherein steps vi through viii are carried out at the same temperature.
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Abstract
Methods for isothermal amplification of nucleic acid by the means of a solid support are disclosed. These methods are useful for applications needing high throughput, in particular nucleic acid sequencing.
87 Citations
27 Claims
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1. A method for isothermally amplifying single stranded nucleic acid molecules immobilized on a solid support comprising:
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i) providing a plurality of different nucleic acid template molecules and attaching a sequence Y to a 5′
end of each of the nucleic acid template molecules and a sequence Z to a 3′
end of each of the nucleic acid template molecules to generate different nucleic acid template molecules comprising the same sequence Y at the 5′
end and the same sequence Z at the 3′
end;ii) providing a solid surface comprising a plurality of first primers comprising sequence X immobilized at their 5′
ends and a plurality of second primers comprising sequence Y immobilized at their 5′
ends, wherein sequence X is hybridizable to sequence Z;iii) contacting the nucleic acid template molecules comprising sequence Y at the 5′
end and sequence Z at the 3′
end with the solid surface to generate at least one 5′
-end immobilized first single stranded nucleic acid template molecule comprising the sequence Y at the 5′
end and the sequence Z at the 3′
end;iv) annealing said at least one 5′
-end immobilized first single stranded nucleic acid template molecule to said first immobilized primers, wherein sequence Z of each template molecule is annealed to one of said first immobilized primers comprising sequence X;v) performing a primer extension reaction using said first immobilized primers that are annealed to said 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;vi) denaturing said double stranded nucleic acid molecules to generate 5′
-end immobilized first and second single stranded nucleic acid molecules;vii) annealing said 5′
-end immobilized first and second single stranded nucleic acid molecules to said first immobilized primers comprising sequence X and said second immobilized primers comprising sequence Y, respectively;viii) 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;ix) repeating steps vi through viii to generate DNA colonies, wherein each DNA colony comprises multiple copies of the 5′
-end immobilized first and second single stranded nucleic acid molecules on said solid surface, wherein steps vi through viii 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, 19, 20, 21, 22, 23, 24, 25)
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26. A method for amplifying nucleic acid molecules comprising:
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(a) providing a plurality of different nucleic acid molecules each comprising a different nucleic acid template sequence, the same sequence Y at the 5′
end of the template sequence and the same sequence Z at the 3′
end of the template sequence;(b) providing a solid surface comprising two different colony primers X′ and
X″
, wherein the colony primers X′
comprise a sequence complementary to the sequence Z and colony primers X″
comprises a sequence that is the same as the sequence Y;(c) immobilizing the different nucleic acid molecules to the solid surface; and (d) amplifying to extend the two different colony primers X′ and
X″
immobilized on the solid surface, wherein the amplifying is carried out isothermally, thereby forming multiple double stranded copies of each of the different nucleic acid template sequences that are immobilized to the surface at both 5′
ends. - View Dependent Claims (27)
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Specification