Process for amplifying nucleic acid

A process for amplifying a target nucleic acid sequence in a double-stranded template nucleic acid, which comprises the steps of:

• (a) annealing first and second primers to first and second template nucleic acids of a double-stranded template nucleic acid, respectively, and synthesizing first and second complementary nucleic acids comprising the complementary sequence of said target nucleic acid by a primer extension reaction, respectively,
• wherein the first 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 in the first strand of the double-stranded template nucleic acid, 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 said 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 first region flanked by said sequences (A) and (B) on the target nucleic acid sequence,
• 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,
• wherein the second primer comprises in its 3′-end portion a sequence (Cc′) that hybridizes to a sequence (C) in the 3′-end portion of the target nucleic acid sequence in the second strand of the double-stranded template nucleic acid, and in the 5′-side of said sequence (Cc′) a sequence (D′) that hybridizes to the complementary sequence (Dc) of a sequence (D) positioned in the 5′-side of said sequence (C) on said target nucleic acid sequence,
• wherein in the absence of an intervening sequence between said sequences (Cc′) and (D′), 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 (Cc′), and Y denotes the number of bases in a second region flanked by said sequences (C) and (D) on the target nucleic acid sequence, and
• wherein in the presence of an intervening sequence between said sequences (Cc′) and (D′), 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 sequences (B′) and (D′) positioned in the 5′-side of the first and second complementary nucleic acids synthesized in step (a) with the sequences (Bc) and (Dc) on the same complementary nucleic acid, respectively, thereby converting the portions of said sequences (A) and (C) on the first and second template strands, respectively, to single-stranded form, and
• (c) annealing additional primers having the same sequence as said primers used in step (a) to the single-stranded sequence (A) and (C) portions of the first and second template nucleic acids from step (b) and conducting a strand displacement reaction, thereby displacing the first and second complementary nucleic acid synthesized in step (a) by the complementary nucleic acids newly synthesized with said additional primers, and wherein steps (a), (b) and (c) are carried out in an isothermal condition.