Patent ID: 7790418
Filing Date: 2010-09-07
Classification: C12Q

Abstract:
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.