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US 6,410,278 B1Granted
Process for 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.
Claims19
A method of amplifying a nucleic acid comprising:
- A) providing a template having (i) a 3′ end portion comprising a first region located 3′ terminal and a first complementary region which, under suitable conditions, anneal to one another to form a first loop, (ii) a 5′ end portion comprising a second region located 5′ terminal and a second complementary region which, under suitable conditions, anneal to one another to form a second loop, and (iii) a region connecting the 3′ end portion and the 5′ end portion;
- B) extending the 3′ terminal of the template to the 5′ end of the template by means of a polymerase having strand displacement activity, when the first region and first complementary region are annealed to one another to form the first loop, to form a template extension which includes a third region located 3′ terminal and a third complementary region which arc substantially the same as the second complementary region and second region, respectively, and which, under suitable conditions, anneal to one another to form a third loop;
- C) annealing to the first loop of the extended template an oligonucleotide primer comprising at the 3′ terminal a nucleotide sequence complementary to at least part of the first loop and at the 5′ terminal a nucleotide sequence complementary to the first region of the template;
- D) extending the oligonucleotide primer along the extended template, by means of a polymerase having strand displacement activity, to form a new template complementary to the template, thereby displacing the template extension formed during said extending in step B);
- E) Further extending the 3′ terminal of the extended template to the 5′ end of the extended template by means of a polymerase having strand displacement activity, when the third region and the third complementary region are annealed to one another to form the third loop, thereby displacing the new template from the extended template;
- and
- F) repeating steps A)-E) using the new template as the template in step A), thereby amplifying the nucleic acid.
4 more elements
A method comprising:
- A) providing a template having (i) a 3′ end portion comprising a first region located 3′ terminal and a first complementary region which, under suitable conditions, anneal to one another to form a first loop, (ii) a 5′ end portion comprising a second region located 5′ terminal and a second complementary region which, under suitable conditions, anneal to one another to form a second loop, and (iii) a region connecting the 3′ end portion and the 5′ end portion;
- B) extending the 3′ terminal of the template to the 5′ end of the template by means of a polymerase having strand displacement activity, when the first region and first complementary region are annealed to one another to form the first loop, to form a template extension which includes a third region located 3′ terminal and a third complementary region that are substantially the same as the second complementary region and second region, respectively, and which, under suitable conditions, anneal to one another to form a third loop;
- C) annealing to the first loop of the extended template an oligonucleotide primer comprising at the 3′ terminal a nucleotide sequence complementary to at least a part of the first loop and at the 5′ terminal a nucleotide sequence complementary to the first region of the template;
- D) extending the oligonucleotide primer along the extended template, by means of a polymerase having strand displacement activity, to form a new template complementary to the template;
- and
- E) displacing the new template from the extended template.
3 more elements
Dependencies are inferred from the claim text.