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.

1 . A kit used for a method for amplifying a nucleic acid, wherein said kit is in a packaged form and contains instructions that direct the use of a DNA polymerase and an endonuclease and said 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.    
     
     
         2 . The kit according to  claim 1 , which further contains at least one selected from the group consisting of: 
 (a) a DNA polymerase having a strand displacement activity;    (b) an endonuclease; and    (c) a buffer.    
     
     
         3 . A kit used for the method for detecting a target nucleic acid, wherein said kit is in a packaged form and contains instructions that direct the use of a DNA polymerase and an endonuclease in a strand displacement reaction and wherein said method comprises: 
 (a) amplifying a target nucleic acid by 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 incubating the reaction mixture for a sufficient time to generate a reaction product; and    (b) detecting the target nucleic acid amplified in step (a).    
     
     
         4 . The kit according to  claim 3 , which contains at least one selected from the group consisting of: 
 (a) a DNA polymerase having a strand displacement activity;    (b) an endonuclease; and    (c) a buffer.    
     
     
         5 . A composition for amplifying a nucleic acid used for a method for amplifying a nucleic acid, which contains: 
 an endonuclease; and    a DNA polymerase having a strand displacement activity, wherein said 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.    
     
     
         6 . The composition according to  claim 5 , which further contains at least one primer that is substantially complementary to a nucleotide sequence of a nucleic acid as a template, wherein the primer is a chimeric oligonucleotide primer that contains a ribonucleotide as well as at least one selected from the group consisitng of a deoxyribonucleotide and a nucleotide analog, the ribonucleotide being positioned at the 3′-terminus or on the 3′-terminal side of the primer.  
     
     
         7 . The composition according to  claim 5 , which contains a buffering component suitable for a nucleic acid amplification reaction.  
     
     
         8 . A composition for amplifying a nucleic acid which contains: 
 (a) at least one primer that is substantially complementary to a nucleotide sequence of a nucleic acid as a template, wherein the primer is a chimeric oligonucleotide primer that contains a ribonucleotide as well as at least one selected from the group consisitng 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) an endonuclease; and    (c) a DNA polymerase having a strand displacement activity.    
     
     
         9 . A composition for amplifying a nucleic acid which contains: 
 (a) at least two primers that are substantially complementary to nucleotide sequences of respective strands of a double-stranded nucleic acid as a template, wherein each primer is a chimeric oligonucleotide primer that 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;    (b) an endonuclease; and    (c) a DNA polymerase having a strand displacement activity.    
     
     
         10 . A composition for amplifying a nucleic acid obtained 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, 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.  
     
     
         11 . A composition for amplifying a nucleic acid obtained by mixing a nucleic acid as a template, a deoxyribonucleotide triphosphate, a DNA polymerase having a strand displacement activity, at least two primers and an endonuclease, wherein each primer is a chimeric oligonucleotide primer that is substantially complementary to the nucleotide sequence of each strand of the double-stranded 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.  
     
     
         12 . A chimeric oligonucleotide primer used for a method for amplifying a nucleotide sequence, which 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, wherein said 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.    
     
     
         13 . A chimeric oligonucleotide primer used for a method for detecting a target nucleic acid, which 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, wherein said method comprises: 
 (a) amplifying a target nucleic acid by 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 incubating the reaction mixture for a sufficient time to generate a reaction product; and    (b) detecting the target nucleic acid amplified in step (a).    
     
     
         14 . The chimeric oligonucleotide primer according to  claim 13 , which is a chimeric oligonucleotide primer for detecting a pathogenic microorganism or a disease-related gene.  
     
     
         15 . The chimeric oligonucleotide primer according to  claim 14 , wherein the pathogenic microorganism is enterohemorrhagic  Escherichia coli, Clostridium botulinum, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia, papilloma  virus, hepatitis C virus or a viroid.  
     
     
         16 . A method for producing a material having an immobilized nucleic acid in which the nucleic acid is arrayed in a predefined region, characterized in that the method comprises: 
 (a) amplifying a nucleic acid to be immobilized by 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 incubating the reaction mixture for a sufficient time to generate a reaction product; and    (b) arraying and immobilizing the nucleic acid amplified in step (a) in a predefined region on a substrate.    
     
     
         17 . The method according to  claim 16 , wherein a single-stranded nucleic acid substantially free of a complementary strand thereto is amplified, and arrayed and immobilized in the predefined region on the substrate.  
     
     
         18 . A material having an immobilized nucleic acid in which the nucleic acid is arrayed in a predefined region produced by the method defined by  claim 16 .  
     
     
         19 . The material according to  claim 18 , wherein a single-stranded nucleic acid is arrayed and immobilized in the predefined region.  
     
     
         20 . The material according to  claim 19 , in which a single-stranded nucleic acid substantially free of a complementary strand thereto is arrayed and immobilized in the predefined region.  
     
     
         21 . A method for detecting a target nucleic acid in a sample, characterized in that the method comprises: 
 (a) preparing a nucleic acid sample suspected to contain a target nucleic acid from a sample;    (b) contacting the nucleic acid sample with the material defined by  claim 18;  and    (c) detecting the target nucleic acid in the nucleic acid sample that hybridizes with the nucleic acid on the material.    
     
     
         22 . A product of a reagent for amplifying a nucleic acid consisting of a packing material and a reagent for amplifying a nucleic acid enclosed in the packing material, wherein the reagent for amplifying a nucleic acid contains a DNA polymerase having a strand displacement activity and/or an RNase H, and description that the reagent for amplifying a nucleic acid can be used for nucleic acid amplification under isothermal conditions is indicated in a label stuck to the packaging material or instructions attached to the packaging material.  
     
     
         23 . A product of a reagent for amplifying a nucleic acid consisting of a packing material and a reagent for amplifying a nucleic acid enclosed in the packing material, wherein the reagent for amplifying a nucleic acid contains a DNA polymerase having a strand displacement activity and/or an endonuclease, and description that the reagent for amplifying a nucleic acid can be used for nucleic acid amplification under isothermal conditions is indicated in a label stuck to the packaging material or instructions attached to the packaging material.