US 2005/0239100 A1 · Method for amplifying nucleic acid sequence

Abstract

A convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

Claims72

Dependent claims

A method for amplifying a nucleic acid, characterized in that the method comprises:

(a) preparing a reaction mixture by mixing a nucleic acid as a template, a deoxyribonucleotide triphosphate, a DNA polymerase having a strand displacement activity, at least one primer and an endonuclease that cleaves an extended strand generated from the primer, wherein the primer is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of the nucleic acid as the template and contains a ribonucleotide as well as at least one selected from the group consisting of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
and
(b) incubating the reaction mixture for a sufficient time to generate a reaction product under conditions where specific annealing of the primer to the nucleic acid as the template, an extended strand synthesis reaction and a strand displacement reaction by the DNA polymerase, and a reaction of cleaving the extended strand by the endonuclease take place.

A method for amplifying a nucleotide sequence using at least two primers, characterized in that the method comprises:

(a) treating a nucleic acid as a template with at least one primer that is substantially complementary to the nucleotide sequence of the nucleic acid and a DNA polymerase to synthesize a primer-extended strand that is complementary to the template, wherein the primer is a chimeric oligonucleotide primer containing a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(b) cleaving the primer-extended strand of a double-stranded nucleic acid obtained in step (a) with an endonuclease at a site that contains the ribonucleotide;
(c) extending a nucleotide sequence that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (b) to effect a strand displacement, wherein a double-stranded nucleic acid containing a regenerated primer-extended strand is reused in step (b);
(d) treating a released displaced strand obtained in step (c) as a template with at least one primer that is different from that used in step (a) and a DNA polymerase to synthesize a primer-extended strand that is complementary to the displaced strand, wherein the primer that is different from that used in step (a) is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of the displaced strand and contains a deoxyribonucleotide and a ribonucleotide, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(e) cleaving the primer-extended strand of a double-stranded nucleic acid obtained in step (d) with an endonuclease at a site that contains the ribonucleotide;
and
(f) extending a nucleotide sequence that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (e) to effect a strand displacement, wherein a double-stranded nucleic acid containing a regenerated primer-extended strand is reused in step (e).

4 more elements

A method for amplifying a nucleic acid using at least two primers, characterized in that the method comprises:

(a) treating a nucleic acid as a template with at least one primer that is substantially complementary to the nucleotide sequence of the nucleic acid and a DNA polymerase to synthesize a primer-extended strand that is complementary to the template, wherein the primer is a chimeric oligonucleotide primer containing a ribonucleotide as well as at least one selected from the group consisting of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(b) extending a nucleic acid that is complementary to the double-stranded nucleic acid as a template obtained in the previous step using a DNA polymerase having a strand displacement activity in the presence of an RNase H to effect a strand displacement and synthesize a displaced strand and a double-stranded nucleic acid;
(c) reusing in step (b) the double-stranded nucleic acid obtained in step (b) as a template;
(d) treating a displaced strand obtained in step (b) as a template with at least one primer that is different from that used in step (a) and a DNA polymerase to synthesize a primer-extended strand that is complementary to the displaced strand, wherein the primer that is different from that used in step (a) is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of the displaced strand and contains a ribonucleotide as well as at least one selected from the group consisting of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(e) extending a nucleic acid that is complementary to the double-stranded nucleic acid as a template obtained in the previous step using a DNA polymerase having a strand displacement activity in the presence of an RNase H to effect a strand displacement and synthesize a displaced strand and a double-stranded nucleic acid;
and
(f) reusing in step (e) the double-stranded nucleic acid obtained in step (e) as a template.

4 more elements

A method for amplifying a nucleic acid, characterized in that the method comprises:

(a) treating a double-stranded nucleic acid as a template with two primers that are substantially complementary to the nucleotide sequences of the respective strands of the double-stranded nucleic acid and a DNA polymerase having a strand displacement activity to synthesize primer-extended strands that are complementary to the template and obtain a double-stranded nucleic acid consisting of the synthesized primer-extended strands being annealed each other, wherein each primer is a chimeric oligonucleotide primer containing a ribonucleotide as well as at least one selected from the group consisting of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(b) cleaving the sites that contain the ribonucleotide of the double-stranded nucleic acid consisting of the primer-extended strands obtained in step (a) with the endonuclease;
(c) extending nucleic acids that are complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-termini of the respective primer portions of the double-stranded nucleic acid in which the primer-extended strands are cleaved obtained in step (b) to effect strand displacements and obtain a double-stranded nucleic acid consisting of the primer-extended strands being annealed each other and a double-stranded nucleic acid consisting of the templates being annealed each other to which the two primers in step (a) are annealed;
(d) extending nucleic acids that are complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-termini of the respective primer portions of the double-stranded nucleic acid to which the two primers are annealed obtained in step (c) to effect strand displacements and obtain a double-stranded nucleic acid consisting of the primer-extended strands being annealed each other and a double-stranded nucleic acid consisting of the templates being annealed each other to which the two primers in step (a) are annealed;
and
(e) reusing in step (d) the double-stranded nucleic acid to which the two primers are annealed obtained in step (d).

3 more elements

A method for amplifying a nucleic acid, characterized in that the method comprises:

(a) treating a double-stranded nucleic acid as a template with two primers that are substantially complementary to the nucleotide sequences of the respective strands of the double-stranded nucleic acid and a DNA polymerase having a strand displacement activity to synthesize primer-extended strands that are complementary to the template and obtain a double-stranded nucleic acid consisting of the synthesized primer-extended strands being annealed each other, wherein each primer is a chimeric oligonucleotide primer containing a ribonucleotide as well as at least one selected from the group consisting of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer;
(b) cleaving the sites that contain the ribonucleotide of the double-stranded nucleic acid consisting of the primer-extended strands obtained in step (a) with the endonuclease;
(c) extending nucleic acids that are complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-termini of the respective primer portions of the double-stranded nucleic acid in which the primer-extended strands are cleaved obtained in step (b) to effect strand displacements and obtain a double-stranded nucleic acid consisting of the primer-extended strands being annealed each other and a double-stranded nucleic acid consisting of the templates being annealed each other to which the two primers in step (a) are annealed;
(d) extending nucleic acids that are complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-termini of the respective primer portions of the double-stranded nucleic acid to which the two primers are annealed obtained in step (c) to effect strand displacements and obtain a double-stranded nucleic acid consisting of the template and the primer-extended strand;
(e) cleaving the sites that contain the ribonucleotide of the double-stranded nucleic acid consisting of the template and the primer-extended strand obtained in step (d) with the endonuclease;
and
(f) extending a nucleic acid that is complementary to the template using a DNA polymerase having a strand displacement activity from the 3′-terminus of the primer portion of the double-stranded nucleic acid in which the primer-extended strand is cleaved obtained in step (e) to synthesize a displaced strand.

4 more elements

Dependencies are inferred from the claim text.

Method for amplifying nucleic acid sequence

Life of this patent

Method for amplifying nucleic acid sequence

Life of this patent

Abstract

Claims72