Primer set for isothermal amplication of a target nucleic acid sequence

A nucleic acid synthesis method for isothermally synthesizing a double-stranded nucleic acid composed of a single-stranded nucleic acid in which the order of at least two different sequences is repeated to a total of two or more times and a nucleic acid complementary to the single-stranded nucleic acid, the nucleic acid synthesis method comprising the following steps (A1) to (A6):

• (A1) providing a single-stranded template nucleic acid having a stem-loop structure in which a 3′ side stem sequence comprising the 3′ end and a 5′ side stem sequence comprising the 5′ end are linked to each other via a loop sequence, with a folding sequence comprising, on the same strand, two sequences that hybridize to each other being linked to the 3′ end of the 3′ side stem sequence;
• (A2) hybridizing a primer to the loop of the single-stranded template nucleic acid and extending the primer toward the 5′ end of the 5′ side stem sequence;
• (A3) successively continuing the extension of the primer that has reached the 5′ end of the 5′ side stem sequence from the 5′ end of the 5′ side stem sequence to the 3′ end of the folding sequence;
• (A4) successively continuing the extension of the primer that has reached the 3′ end of the folding sequence in the step (A3) toward the 5′ end of the 5′ side stem sequence again, and, by the continuing primer extension, rendering the primer-extended strand hybridizing to the single-stranded template nucleic acid formed in the step (A2) single-stranded through a strand displacement reaction;
• (A5) terminating the extension of the primer in the step (A4) at the 5′ end of the 5′ side stem sequence;
• and
• (A6) extending the 3′ end of the folding sequence in the single-stranded template nucleic acid using the primer-extended strand that has been rendered single-stranded in the step (A4) as a template.

A nucleic acid synthesis method for isothermally synthesizing a double-stranded nucleic acid composed of a single-stranded nucleic acid in which the order of at least two different sequences is repeated to a total of two or more times and a nucleic acid complementary to the single-stranded nucleic acid, the nucleic acid synthesis method comprising at least one of:

• a first reaction step;
• and
• a second reaction step,
• the first reaction step comprising the following steps (B1) to (B3):
• (B1) providing two double strands in a state where their sequences are in opposite orientations, the double strands each being composed of a single-stranded nucleic acid that comprises, in a region comprising the 3′ end, a folding sequence comprising, on the same strand, two sequences that hybridize to each other and a single-stranded nucleic acid complementary to the single-stranded nucleic acid;
• (B2) extending, through a strand displacement reaction, the 3′ end of the folding sequence in the single-stranded nucleic acid in one of the two double strands provided in the step (B1) using the complementary single-stranded nucleic acid in the other double strand as a template, thereby forming a partial double strand in which part of the extended strand of the single-stranded nucleic acid in said one of the double strands hybridizes to the complementary single-stranded nucleic acid in the other double strand;
• and
• (B3) extending, in the partial double strand in the step (B2), the 3′ end of the complementary single-stranded nucleic acid using the single-stranded nucleic acid as a template, thereby forming a complete double strand,
• the second reaction step comprising the following steps (C1) to (C3):
• (C1) providing one double strand composed of a single-stranded nucleic acid that comprises, in a region comprising the 3′ end, a folding sequence comprising, on the same strand, two sequences that hybridize to each other and a single-stranded nucleic acid complementary to the single-stranded nucleic acid;
• (C2) extending, through a strand displacement reaction, the 3′ end of the folding sequence in the single-stranded nucleic acid in the double strand provided in the step (C1) using the complementary single-stranded nucleic acid as a template from the 3′ end to the 5′ end of the complementary single-stranded nucleic acid, thereby forming a partial double strand in which part of the extended strand of the single-stranded nucleic acid hybridizes to the complementary single-stranded nucleic acid;
• and
• (C3) extending, in the partial double strand in the step (C2), the 3′ end of the complementary single-stranded nucleic acid using the single-stranded nucleic acid as a template, thereby forming a complete double strand.