Method of assay of target nucleic acid

Abstract

A simple and accurate method for assay of a single-stranded RNA containing a specific nucleic acids sequence in a sample at almost constant temperature by using at least the following reagents (A) to (I), which comprises a step of adding the reagents (A) to (I) one by one (in any order), in combinations of at least two or all at once and a step of measuring a fluorescent signal in the presence of the reagent (I) at least once after addition of at least the reagents (A) to (H); (A) a first single-stranded oligonucleic acid complementary to a sequence neighboring the 5′ end of the specific nucleic acids sequence in the single-stranded RNA, (B) a second single-stranded oligo DNA complementary to a 3′-end sequence within the specific nucleic acids sequence, (C) an RNA-dependent DNA polymerase, (D) deoxyribonucleoside triphosphates, (E) a third single-stranded oligo DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) a 5′-end sequence within the specific nucleic acids sequence, in this order from the 5′ end, (F) a DNA-dependent DNA polymerase, (G) a DNA-dependent RNA polymerase, (H) ribonucleoside triphosphates, and (I) a fourth single-stranded oligo DNA complementary to the specific nucleic acids sequence which is labeled so that it gives off a measurable fluorescent signal on hybridization with a nucleic acid containing the specific nucleic acids sequence.

What is claimed is:  
     
         1 . A simple and accurate method for assay of a single-stranded RNA containing a specific nucleic acids sequence in a sample at almost constant temperature by using at least the following reagents (A) to (I), which comprises  
       a step of adding the reagents (A) to (I) one by one (in any order), in combinations of at least two or all at once and a step of measuring a fluorescent signal in the presence of the reagent (I) at least once after addition of at least the reagents (A) to (H); 
 (A) a first single-stranded oligonucleic acid complementary to a sequence neighboring the 5′ end of the specific nucleic acids sequence in the single-stranded RNA,  
 (B) a second single-stranded oligo DNA complementary to a 3′-end sequence within the specific nucleic acids sequence,  
 (C) an RNA-dependent DNA polymerase,  
 (D) deoxyribonucleoside triphosphates,  
 (E) a third single-stranded oligo DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) a 5′-end sequence within the specific nucleic acids sequence, in this order from the 5′ end,  
 (F) a DNA-dependent DNA polymerase,  
 (G) a DNA-dependent RNA polymerase,  
 (H) ribonucleoside triphosphates, and  
 (I) a fourth single-stranded oligo DNA complementary to the specific nucleic acids sequence which is labeled so that it gives off a measurable fluorescent signal on hybridization with a nucleic acid containing the specific nucleic acids sequence.  
 
     
     
         2 . The method according to    claim 1   , wherein the temperature is selected from the range of from 35 to 60° C.  
     
     
         3 . The method according to    claim 1   , wherein the first oligonucleic acid as the reagent (A) is a DNA, and the method further comprises a step of adding an RNaseH and a subsequent step of deactivating the RNaseH by heating or by addition of an inhibitor prior to addition of the reagent (B).  
     
     
         4 . The method according to    claim 3   , wherein addition of the reagent (A) is followed by simultaneous addition of the reagents (B) to (H), and further by addition of the reagent (I).  
     
     
         5 . The method according to    claim 3   , wherein addition of the reagent (A) is followed by simultaneous addition of the reagents (B) to (I).  
     
     
         6 . The method according to    claim 1   , wherein the first oligonucleic acid as the reagent (A) is a ribozyme or a DNAzyme.  
     
     
         7 . The method according to    claim 1   , which further uses dimethyl sulfoxide and/or an enzyme which degrades RNA in a DNA-RNA double strand.  
     
     
         8 . The method according to    claim 7   , which uses dimethyl sulfoxide at a concentration of from 5 to 20%.  
     
     
         9 . The method according to    claim 7   , wherein the enzyme which degrades RNA in a DNA-RNA double strand is the RNA-dependent DNA polymerase as the reagent (C).  
     
     
         10 . The method according to    claim 1   , wherein an enzyme having both an RNA-dependent DNA polymerase activity and a DNA-dependent DNA polymerase activity is used as the reagents (C) and (F) to virtually omit addition of the reagent (C) or the reagent (F).  
     
     
         11 . The method according to    claim 10   , wherein the enzyme is avian myoblastome virus polymerase.  
     
     
         12 . The method according to    claim 1   , wherein the second and third oligo DNAs as the reagents (B) and (E) are used at concentrations of from 0.02 to 1 μM.  
     
     
         13 . The method according to    claim 1   , wherein the DNA-dependent RNA polymerase as the reagent (G) is at least one enzyme selected from the group consisting of phage SP6 polymerase, phage T3 polymerase and phase T7 polymerase.  
     
     
         14 . The method according to    claim 1   , wherein the fourth oligo DNA as the reagent (I) is a DNA which is linked to a fluorescent intercalative dye so that the fluorescent intercalative dye changes its fluorescence characteristic on hybridization of the DNA with another nucleic acid by intercalating into the resulting double strand.  
     
     
         15 . The method according to    claim 1    or    14   , wherein the fourth oligo DNA as the reagent (I) is a DNA which has a 3′-end sequence uncomplementary to the specific nucleic acids sequence or has a modified 3′ end.  
     
     
         16 . The method according to    claim 1   , which further comprises a step of detecting or quantifying the single-stranded RNA in the sample based on the measured fluorescent signal or change in the measured fluorescent signal.  
     
     
         17 . The method according to    claim 1   , wherein all the reagents are chloride-free.  
     
     
         18 . The method according to    claim 1   , which further uses an acetate.  
     
     
         19 . The method according to    claim 18   , wherein the acetate is magnesium acetate at a concentration of from 5 to 20 mM or potassium acetate at a concentration of from 50 to 200 mM.  
     
     
         20 . The method according to    claim 1   , which further uses sorbitol.  
     
     
         21 . A simple method for producing a nucleic acid having a specific nucleic acids sequence at almost constant temperature by using at least the following reagents (A) to (H), which comprises a step of adding the reagents (A) to (G) one by one (in any order), in combinations of at least two or all at once to a single-stranded DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) the specific nucleic acids sequence, in this order from the 5′ end or to a double-stranded DNA consisting of the single-stranded DNA and a complementary DNA strand and a step of measuring a fluorescent signal from the reagent (H) at least once after addition of at least the reagents (A) to (G); 
 (A) a single-stranded oligo DNA complementary to a 3′-end sequence within the specific nucleic acids sequence,  
 (B) an RNA-dependent DNA polymerase,  
 (C) a DNA-dependent DNA polymerase,  
 (D) deoxyribonucleoside triphosphates,  
 (E) a DNA-dependent RNA polymerase,  
 (F) ribonucleoside triphosphates,  
 (G) a single-stranded DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) a 5′-end sequence within the specific nucleic acids sequence, in this order from the 5′ end,  
 (H) a fourth single-stranded labeled oligo DNA complementary to the specific nucleic acids sequence which gives a measurable fluorescent signal on hybridization with a nucleic acid containing the specific nucleic acids sequence.  
 
     
     
         22 . The method for producing a single-stranded RNA having a specific nucleic acids sequence according to    claim 21   , wherein a DNase is added when the measured fluorescent signal or change in the measured fluorescent signal indicates production of a predetermined amount of the specific nucleic acids sequence.  
     
     
         23 . The method for producing a double-stranded DNA consisting of a DNA strand having a specific nucleic acids sequence and a complementary DNA strand according to    claim 21   , wherein an RNase is added when the measured fluorescent signal or change in the measured fluorescent signal indicates production of a predetermined amount of the specific nucleic acids sequence.  
     
     
         24 . A reagent set for performing the method according to    claim 1    or    21   , which comprises at least 
 a first reagent containing the first single-stranded oligonucleic acid,  
 a second reagent containing tris-acetate, magnesium acetate, potassium acetate, sorbitol and dimethyl sulfoxide,  
 a third reagent containing dithiothreitol, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, bovine serum albumin, the second single-stranded oligo DNA and the third single-stranded oligo DNA,  
 a fourth reagent containing an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and an RNase inhibitor and  
 a fifth reagent containing the fourth single-stranded oligo DNA.  
 
     
     
         25 . A reagent set for performing the method according to    claim 1    or    21   , which comprises at least 
 a first reagent containing the first single-stranded oligonucleic acid,  
 a second reagent containing tris-acetate, magnesium acetate, potassium acetate, sorbitol and dimethyl sulfoxide,  
 a third reagent containing dithiothreitol, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, bovine serum albumin, the second single-stranded oligo DNA, the third single-stranded oligo DNA and the fourth single-stranded oligo DNA and  
 a fourth reagent containing an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and an RNase inhibitor.  
 
     
     
         26 . A reagent set for performing the method according to    claim 1    or    21   , which comprises at least 
 a first reagent containing the first single-stranded oligonucleic acid,  
 a second reagent containing tris-acetate, magnesium acetate, potassium acetate, sorbitol and dimethyl sulfoxide,  
 a third reagent containing dithiothreitol, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, bovine serum albumin, the second single-stranded oligo DNA and the third single-stranded oligo DNA,  
 a fourth reagent containing the fourth single-stranded oligo DNA, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and an RNase inhibitor.  
 
     
     
         27 . A reagent for performing the method according to    claim 1    or    21   , which comprises at least the first single-stranded oligonucleic acid, the second single-stranded oligo DNA, the third single-stranded oligo DNA, the fourth single-stranded oligo DNA, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, tris-acetate, magnesium acetate, potassium acetate, sorbitol, dimethyl sulfoxide, dithiothreitol, bovine serum albumin and an RNase inhibitor.  
     
     
         28 . The reagent set or reagent according to any one of    claims 24    to    27   , wherein an enzyme having both an RNA-dependent DNA polymerase activity and a DNA-dependent DNA polymerase activity is used at least as the RNA-dependent DNA polymerase and as the DNA-dependent DNA polymerase.