Patent ID: 7803579
Filing Date: 2010-09-28
Classification: C12Q

Abstract:
1. A process for synthesizing a nucleic acid complementary to a target nucleic acid sequence in a template nucleic acid, which comprises the steps of: (a) annealing a primer to said template nucleic acid and synthesizing a complementary nucleic acid comprising the complementary sequence of said target nucleic acid sequence by a primer extension reaction, wherein the primer comprises in its 3′-end portion a sequence (Ac′) that hybridizes to a sequence (A) in the 3′-end portion of the target nucleic acid sequence, and in the 5′-side of said sequence (Ac′), a sequence (B′) that hybridizes to the complementary sequence (Bc) of a sequence (B) positioned in the 5′-side of said sequence (A) on the target nucleic acid sequence, wherein in the absence of an intervening sequence between said sequences (Ac′) and (B′), X is in the range of 10 to 30, (X−Y)/X is in the range of −1.00 to 0.75, and (X+Y) is in the range of 30 to 50, in which X denotes the number of bases in said sequence (Ac′), and Y denotes the number of bases in a region flanked by said sequences (A) and (B) on the target nucleic acid sequence, and wherein in the presence of an intervening sequence between said sequences (Ac′) and (B′), X is in the range of 10 to 30, {X−(Y−Y′)}/X is in the range of −1.00 to 0.75, and (X+Y+Y′) is in the range of 30 to 50, in which X and Y have the same meanings as above, and Y′ denotes the number of bases in said intervening sequence; (b) hybridizing the sequence (B′) positioned in the 5′-side of the complementary nucleic acid synthesized in step (a) with the sequence (Bc) on the same complementary nucleic acid, thereby allowing the portion of said sequence (A) on the template nucleic acid to be single-stranded, and (c) annealing another primer having the same sequence as the primer used in step (a) to the single-stranded sequence (A) portion of the template nucleic acid from step (b) and conducting a strand displacement reaction, thereby displacing the complementary nucleic acid synthesized in step (a) by the complementary nucleic acid newly synthesized with said another primer, and wherein steps (a), (b) and (c) are carried out in an isothermal condition.