Method of synthesizing nucleic acid

Abstract

An oligonucleotide having a novel structure and a method of synthesizing nucleic acid uses 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 method synthesizes highly specific nucleic acid on the basis of this method of synthesizing nucleic acid.

1 . 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 and a region R3 and a region R2 and a region R1 in this order from the 5′-side of the template, the primer set comprising:
 a first oligonucleotide 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 comprising another region R2 having the same nucleotide sequence as the region R2 that is complementary to a region R2c and another region R1c linked to the 5′-side of the region R2 and having a nucleotide sequence complementary to the region R1; 
 a third oligonucleotide comprising a region F3 having a nucleotide sequence complementary to the region F3c; and 
 a fourth oligonucleotide comprising another region R3 having a nucleotide sequence as the region R3 that is complementary to a region R3c linked to the 3′-side of the region R2c in the complementary chain synthesized with the first oligonucleotide. 
 
     
     
         2 . The primer set of  claim 1 , wherein a distance between the region F2c and the region F1c is 0 to 100 bases, and
 a distance between the region R2c and the region R1c is 0 to 100 bases.   
     
     
         3 . The primer set of  claim 1 , wherein a distance between the region F2c and the region F1c is 10 to 70 bases, and
 a distance between the region R2c and the region R1c is 10 to 70 bases.   
     
     
         4 . The primer set of  claim 2 , wherein the distance between the region F2c and the region F3 is 0 base and the distance between the region R2c and the region R3 is 0 bases. 
     
     
         5 . The primer set of  claim 3 , wherein the distance between the region F2c and the region F3 is 0 bases and the distance between the region R2c and the region R3 is 0 bases. 
     
     
         6 . The primer set of  claim 1 , wherein the complementary chain synthesized with the first oligonucleotide based on the template comprises the another region F1c at the 5′ terminal thereof and a region F1 that is complementary to the another region F1c, a complementary chain synthesized with the second oligonucleotide based on the complementary chain synthesized with the first oligonucleotide comprises another region F1 at the ′3 terminal thereof and yet another region F1c that is complementary to the another region F1, a complementary chain synthesized with the third oligonucleotide displaces the complementary chain synthesized with the first oligonucleotide from the template, and a complementary chain synthesized with the fourth oligonucleotide displaces the complementary chain synthesized with the second oligonucleotide from the complementary chain synthesized with the first oligonucleotide. 
     
     
         7 . 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 and a region R3 and a region R2 and a region R1c in this order from the 5′-side of the template, the kit comprising:
 a first oligonucleotide 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 comprising another region R2 having the same nucleotide sequence as the region R2 that is complementary to a region R2c and another region R1c linked to the 5′-side of the region R2 and having a nucleotide sequence complementary to the region R1; 
 a third oligonucleotide comprising a region F3 having a nucleotide sequence complementary to the region F3c; 
 a fourth oligonucleotide comprising another region R3 having the same nucleotide sequence as the region R3 that is complementary to a region R3c linked to the 3′-side of the region R2c in the complementary chain synthesized with the first oligonucleotide; 
 a DNA polymerase catalyzing a strand displacement-type reaction; and 
 a nucleotide serving as a substrate for the DNA polymerase. 
 
     
     
         8 . The kit of  claim 7 , wherein a distance between the region F2c and the region F1c located at the 5′-side of F2c is 0 to 100 bases, and
 a distance between the region R2c and the region R1c located at the 5′-side of R2c is 0 to 100 bases. 
 
     
     
         9 . The kit of  claim 7 , wherein a distance between the region F2c and the region F1c is 10 to 70 bases, and
 a distance between the region R2c and the region R1c is 10 to 70 bases.   
     
     
         10 . The kit according to  claim 8 , wherein the distance between the region F2c and the region F3 is 0 bases and the distance between the region R2c and the region R3 is 0 bases. 
     
     
         11 . The kit according to  claim 9 , wherein the distance between the region F2c and the region F3 is 0 bases and the distance between the region R2c and the region R3 is 0 bases. 
     
     
         12 . The kit according to  claim 7 , wherein the complementary chain synthesized with the first oligonucleotide based on the template comprises the another region F1c at the 5′ terminal thereof and a region F1 that is complementary to the another region F1c, a complementary chain synthesized with the second oligonucleotide based on the complementary chain synthesized with the first oligonucleotide comprises another region F1 at the ′3 terminal thereof and yet another region F1c that is complementary to the another region F1, a complementary chain synthesized with the third oligonucleotide displaces the complementary chain synthesized with the first oligonucleotide from the template, and a complementary chain synthesized with the fourth oligonucleotide displaces the complementary chain synthesized with the second oligonucleotide from the complementary chain synthesized with the first oligonucleotide.