Method of synthesizing nucleic acid
The present invention relates to an oligonucleotide having a novel structure and a method of synthesizing nucleic acid by using the same as a primer. This oligonucleotide is provided at the 5′-side of the primer with a nucleotide sequence substantially the same as a region synthesized with this primer as the origin of synthesis. The present invention realizes synthesis of nucleic acid based on an isothermal reaction with a simple constitution of reagents. Further, the present invention provides a method of synthesizing highly specific nucleic acid on the basis of this method of synthesizing nucleic acid.
1. A kit designed for synthesizing nucleic acid by amplifying a template nucleic acid comprising a region F3c, a region F2c and a region F1c in this order from the 3′-side of the template, a region R3 and a region R2 in this order from the 5′-side of the template, and a region R1 linked to the 3′-side of the region R2, the kit comprising:
a first oligonucleotide having 20 to 200 base pairs and comprising a region F2 having a nucleotide sequence complementary to the region F2c and another region F1c linked to the 5′-side of the region F2 and having the same nucleotide sequence as the region F1c;
a second oligonucleotide having 20 to 200 base pairs and comprising another region R2 having the same nucleotide sequence as the region R2 and a region R1c having a nucleotide sequence complementary to the region R1 and linked to the 5′-side of the another R2 region;
a third oligonucleotide having 10 to 200 base pairs and comprising a region F3 having a nucleotide sequence complementary to the region F3c; and
a fourth oligonucleotide having 10 to 200 base pairs and comprising another region R3;
a DNA polymerase catalyzing a strand displacement-type reaction; and
a nucleotide serving as a substrate for the DNA polymerase;
wherein said kit does not include a restriction enzyme and wherein each of the regions is at least 10 bases long.
2. The kit according to claim 1 , further comprising a detector for detection of a product of nucleic acid synthetic reaction.
3. A primer set designed for synthesizing nucleic acid by amplifying a template nucleic acid comprising a region F3c, a region F2c and a region F1c in this order from the 3′-side of the template, a region R3 and a region R2 in this order from the 5′-side of the template and a region R1 linked to the 3′-side of the region R2, the set comprising:
a first oligonucleotide having 20 to 200 base pairs and comprising a region F2 having a nucleotide sequence complementary to the region F2c and another region F1c linked to the 5′-side of the region F2 and having the same nucleotide sequence as the region F1c;
a second oligonucleotide having 20 to 200 base pairs and comprising another R2 having the same nucleotide sequence as the region R2 and a region R1c having a nucleotide sequence complementary to the region R1 and linked to the 5′-side of the another R2 region;
a third oligonucleotide having 10 to 200 base pairs and comprising a region F3 having a nucleotide sequence complementary to the region F3c; and
a fourth oligonucleotide having 10 to 200 base pairs and comprising another region R3 having the same nucleotide sequence as the region R3;
wherein said primer set does not include a restriction enzyme and wherein each of the regions is at least 10 bases long.
4. The kit according to claim 1 , wherein the second oligonucleotide is free of a promoter recognized by RNA polymerase.
5. The primer set according to claim 3 , wherein the second oligonucleotide is free of a promoter recognized by RNA polymerase.
6. The kit according to claim 1 , wherein the first oligonucleotide is free of a promoter recognized by RNA polymerase, and the kit does not include a restriction enzyme.
7. The primer set according to claim 3 , wherein the first oligonucleotide is free of a promoter recognized by RNA polymerase, and the primer set does not include a restriction enzyme.
8. The kit according to claim 1 , wherein the complementary chain synthesized with the second oligonucleotide comprises another region R1c at the 5′ terminal thereof and another region R1 that is complementary to the another region R1c.