Method of synthesizing nucleic acid
AssigneeEiken Kagaku Kabushiki Kaisha
InventorsTsugunori Notomi, Tetsu Hase
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Abstract
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.
Claims8
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.
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.
Dependencies are inferred from the claim text.