Abstract:
Disclosed is a novel isothermal nucleic acid amplification method enabling inexpensive and simple and easy detection. The method includes introducing nicking enzyme recognition sequences into an analysis target nucleic acid using nicking enzyme recognition sequence-containing primers and isothermally amplifying a specific region of the target nucleic acid using the primers, a nicking enzyme and a DNA polymerase having strand displacement activity.

Description:
CLAIM OF PRIORITY 
       [0001]    The present application claims priority from Japanese application JP 2006-328211 filed on Dec. 5, 2006, the content of which is hereby incorporated by reference into this application. 
       FIELD OF THE INVENTION 
       [0002]    The present invention relates to a nucleic acid (DNA or RNA) amplification method. More particularly, it relates to a novel isothermal nucleic acid amplification method which uses a primer(s) having a nicking enzyme recognition site and a DNA polymerase having strand displacement activity. 
       BACKGROUND OF THE INVENTION 
       [0003]    In studying vital phenomena, the technique of DNA and RNA amplification is used for various purposes. For example, the competitive PCR method (Non-patent document 1: A. Wang, et al., Proc. Natl. Acad. Sci. USA, 86, 9717-9721 (1989)) and the real-time PCR method (Non-patent document 2: K. Edwards, et al., Journal of Antimicrobial Chemotherapy, 54, 968 (2004)), among others, are known as methods for gene expression analysis and gene expression level quantification. These methods all employ the PCR (polymerase chain reaction) technique (Non-patent document 3: R. K. Saiki, et al., Science, 239, 487-491 (1988)), which is now a common nucleic acid amplification method, to determine the gene expression level based on the amount of the gene amplified. 
         [0004]    The nucleic acid amplification methods for the above-mentioned analytical purposes comprise three steps, namely the steps of denaturation of a double-stranded DNA to single-stranded DNAs, annealing of primers to a single-stranded DNA, and complementary strand extension from the primers, or two steps, namely the steps of such denaturation and strand extension, and it is essential to repeat a cycle from a higher temperature step to a lower temperature step. For carrying out such steps, it is necessary to carry out the PCR using a thermal cycler by which exact temperature control is possible. The time required for setting the apparatus and reaction mixture at respective required temperatures increases with the number of cycles and, therefore, much time is required for the intended analysis. 
         [0005]    For solving such problems, nucleic acid amplification methods which can be carried out under isothermal conditions have been developed. For example, the NASBA (nucleic acid sequence-based amplification) method (Non-patent document 4: J. Compton, et al., Nature, 350, 91-92 (1991)), the SDA (strand displacement amplification) method (Non-patent document 5: G. T. Walker et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992)), the 3SR (self-sustained sequence replication) method (Non-patent document 6: J. C. Guatelli, et al., Proc. Natl. Acid. Sci. USA, 87, 1874-1878 (1990)), the TMA (transcription-mediated amplification) method (Patent document 1: JP No. 3241717), the Qβ replicase amplification method (Patent document 2: JP No. 2710159), and the LAMP (loop-mediated isothermal amplification) method (Non-patent document 7: T. Natomi, et al., Nucleic Acids Research, 28, e63 (2000)), among others, are known as principal methods. According to these isothermal nucleic acid amplification methods, the primer extension, the primer annealing to a single-stranded chain extension product, and the subsequent primer extension are realized in a reaction mixture maintained at a constant temperature. 
         [0006]    As for the methods using a DNA polymerase (SDA method and LAMP method) among these isothermal nucleic acid amplification methods, the SDA method uses dATPαS, in lieu of dATP, as a substrate for providing priming sites to serve as the starting points of amplification, and the LAMP method is designed so that the primer extension product may form a self-looped structure. Therefore, the SDA method encounters such problems as a low yield of substrate incorporation by enzyme and the resulting decrease in amplification efficiency, while the LAMP method has problems such that primer designing is difficult and it is difficult to design optimal primers for various test items. An isothermal nucleic acid amplification method by which these problems can be solved is thus demanded. 
         [0007]    On the other hand, a method of synthesizing oligonucleotides, about 15 bases in length, utilizing, as priming sites, nicks formed on one strand of a double-stranded DNA using a nicking enzyme has also been reported (Non-patent document 8: Jeffery Van Ness, et al., Proc. Natl. Acad. Sci. USA, vol. 100, No. 8, 4504-4509 (2003)). However, this method cannot amplify nucleic acids exceeding 20 bases in length. 
       SUMMARY OF THE INVENTION 
       [0008]    Accordingly, it is a primary object of the present invention to provide a method which can solve the problems with the prior art isothermal amplification methods and enables gene expression or gene expression level quantification with ease and at low cost. 
         [0009]    The inventors invented an isothermal amplification method which uses a DNA polymerase having strand displacement activity and a nicking enzyme. The present invention uses primers for amplification having a nicking enzyme recognition sequence-containing anchored sequence on the 5′ terminus side of a sequence recognizing a target sequence. As a result of extension of primers annealed to a target gene, a double-stranded DNA having nicking enzyme recognition sequences at both ends is formed. A nicking enzyme is caused to act on the double-stranded DNA to form a nick resulting from phosphodiester bond cleavage on one strand of the double-stranded DNA. By using the nicks formed as priming sites, it becomes possible to amplify the target sequence. A nick is formed in each nicking enzyme recognition sequence and, therefore, one nick is formed at each end of the double-stranded DNA. When a DNA polymerase and a substrate are allowed to act on these nicks, a DNA strand is synthesized. Owing to the strand displacement activity, the DNA strand extension proceeds isothermally without any thermal denaturation step, and the double-stranded DNA having a nicking enzyme recognition sequence at each end is regenerated. The DNA strand stripped off by the strand displacement activity hybridizes with amplification primers or a complementary DNA thereof to provide novel starting points for DNA synthesis. 
         [0010]    Thus, according to an embodiment of the invention, nicking enzyme recognition sequences are introduced into a double-stranded DNA and nicks are formed on the double-stranded DNA by a nicking enzyme, whereby a DNA strand is caused to extend from the nicks formed for double-stranded DNA synthesis. The steps of nick formation on the double-stranded DNA by the action of the nicking enzyme and DNA strand extension with the nicks formed serving as priming sites are repeated substantially under isothermal conditions, namely at a temperature of 30° C. to 75° C., enabling isothermal amplification of the target sequence. 
         [0011]    According to an embodiment of the invention, a nicking enzyme recognition sequence is introduced into each strand constituting a double-stranded nucleic acid and DNA strand extension is allowed to proceed from each nick site and, therefore, the region between the two nicks is the region to be amplified. The length of a nicking enzyme recognition sequence is generally 5-7 bases and a nick is formed in a phosphodiester bond within the recognition sequence or about 4 bases on the 3′ side from the recognition sequence. Nicking enzyme recognition sequences are introduced using primers having a base sequence about 15-25 bases in length and specifically hybridizing with a target gene, further having an appropriate linker sequence (0-5 bases long) on the 5′ end side according to need, and further having a nicking enzyme recognition sequence (5-7 bases long) on the 5′ end side. Therefore, the site of each nick formed is about 10-30 bases from the 3′ end of each primer. Since a DNA having a nicking enzyme recognition site at each end thereof is formed using such primers as mentioned above, the region between the two nicks is at least 21 bases in length. Therefore, the length of the DNA region amplified according to the present invention is at least 21 bases in length. 
         [0012]    The nucleic acid amplification method of the invention makes it possible to design primers with ease using the ordinary substrate dATP and can realize an inexpensive and simple isothermal nucleic acid amplification method. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  is a flow chart illustrating a first aspect of the invention; 
           [0014]      FIG. 2  is a flow illustrating the process in which the nicking enzyme recognition sequence-containing double-stranded DNA is obtained in the flow chart illustrating the first aspect of the invention; 
           [0015]      FIG. 3  is a flow chart illustrating a second aspect of the invention; 
           [0016]      FIG. 4  is a flow chart illustrating a third aspect of the invention; 
           [0017]      FIG. 5  is an electrophoretic profile of the amplification product obtained in accordance with the flow chart illustrating the first aspect of the invention; and 
           [0018]      FIG. 6  is a graphic representation of the results of real-time monitoring of the amplification process according to the flow chart illustrating a third aspect of the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0019]      FIG. 1  is a flow chart illustrating a first aspect of the present invention. In this aspect, the invention relates to a method of amplifying and detecting a nucleic acid sequence, and the gene amplification method is characterized in that it comprises a first step of using a primer  4  having a sequence  2  having a sequence specific (complementary) to the base sequence of a target gene  1  and a sequence  3  attached to the 5′ end of the sequence  2  and noncomplementary to the base sequence of the target gene  1  and containing a nicking enzyme recognition sequence  3 , a primer  6  capable of hybridizing with the target gene  1  on the side upstream of the primer  4  (on the side reverse to the direction of extension of the primer  4 ) and having a sequence  5  specific (complementary) to a base sequence of the target gene  1 , a primer  14  containing a sequence  12  specific (complementary) to a base sequence of the target gene  1  and a sequence  13  attached to the 5′ end of the sequence  12  and noncomplementary to the base sequence of the target gene and containing a nicking enzyme recognition sequence, and a primer  16  capable of hybridizing with the target gene  1  on the side upstream from the primer  14  (on the side reverse to the direction of extension of the primer  14 ) and having a sequence  15  specific (complementary) to a base sequence of the target gene  1 , together with the target gene  1  as a template, to cause extension of the primer  4 , primer  14 , primer  6 , and primer  16 , a second step of converting the nicking enzyme recognition sequence-containing double-stranded DNA  8  obtained as a result of the first step to a nick-containing double-stranded DNA  9  under the action of a nicking enzyme, and a third step of regenerating the nicking enzyme recognition sequence-containing double-stranded DNA  8  by causing a DNA polymerase having strand displacement activity to act, for DNA strand extension, on the nick-containing double-stranded DNA  9  the nicks of which serve as priming sites. 
         [0020]      FIG. 2  shows in detail how the nicking enzyme recognition sequence-containing double-stranded DNA  8  is formed in the first step shown in the flow chart illustrating the first aspect of the invention. The primer  4  having a sequence  2  having a sequence specific to a base sequence of the target gene  1  and a nicking enzyme recognition sequence-containing sequence  3 , and the primer  14  containing a sequence  12  having a sequence specific to a base sequence of the target gene  1  and a nicking enzyme recognition sequence-containing sequence  13  are extended with the target gene as a template. The primer  6  capable of hybridizing with the target gene on the side upstream from the primer  4  (on the side reverse to the direction of extension of the primer  4 ) and having a sequence  5  specific to a base sequence of the target gene  1  is extended while stripping off the primer  4 -derived extension product  21  obtained in the initial step, and the primer  16  capable of hybridizing with a base sequence of the target gene  1  on the side upstream from the primer  14  (on the side reverse to the direction of extension of the primer  14 ) and having a sequence  15  specific to a base sequence of the target gene  1  is extended while stripping off the primer  14 -derived extension product  31  obtained in the initial step. The primer extension products  21  and  31  hybridize with each other to form a double-stranded DNA  32 , and the extension of the double stranded DNA  32  results in the formation of a nicking enzyme recognition sequence-containing double-stranded DNA  33 . The double-stranded DNA  33  corresponds to the double-stranded DNA  8  in  FIG. 1 ; thus, it is possible to introduce a nicking enzyme recognition sequence in each strand of the target gene. 
         [0021]      FIG. 3  is a flow chart illustrating a second aspect of the present invention. Using a primer  44  containing a sequence  42  having a sequence specific (complementary) to a nicking enzyme recognition sequence-containing target gene  41  and a nicking enzyme recognition sequence-containing sequence  43  attached to the 5′ end of the sequence  42  and noncomplementary to the base sequence of the target gene  41 , a primer  46  capable of hybridizing with the target gene  41  on the side upstream from the primer  44  (on the side reverse to the direction of extension of the primer  44 ) and having a sequence  45  specific (complementary) to a base sequence of the target gene  41 , and a primer  54  containing a sequence  52  specific (complementary) to the target gene  41 , strand extension is carried out with the nicking enzyme recognition sequence-containing target gene  41  as a template. The primer  44  and primer  54  are designed so that they may hybridize with the target gene at respective sites apart on either side of the nicking enzyme recognition sequence  53  in the target gene  41 . 
         [0022]    The primer  46  having a sequence  45  specific (complementary) to the target gene  41  is extended while stripping off the primer  44 -derived extension product  51 . The primer  54  containing a sequence  52  having a sequence specific to the target gene  41  hybridizes with the stripped-off extension product  51 , followed by strand extension to give a double-stranded DNA  55  having a plurality of nicking enzyme recognition sequences. Thereafter, a nicking enzyme is caused to act on the DNA  55 , whereupon a nick-containing double-stranded DNA  54  is formed. The nicks formed serve as priming sites and when DNA strand extension is carried out under the action of a DNA polymerase having strand displacement activity, the nicking enzyme recognition sequence-containing double-stranded DNA  55  is regenerated. A nicking enzyme is again caused to act on the double-stranded DNA  53  to form the nick-containing DNA  54  and the DNA strand extension is carried out under the action of a DNA polymerase having strand displacement activity with the nicks serving as priming sites, whereupon the double-stranded DNA  55  is formed. By repeating these steps, it becomes possible to amplify a specific region of the target gene. 
         [0023]      FIG. 4  is a flow chart illustrating a third aspect of the invention. A target RNA  61  to be amplified is subjected to reverse transcription reaction using a primer  64  having a sequence  62  having a sequence specific (complementary) to the target RNA  61  and further having a nicking enzyme recognition sequence-containing sequence  63  attached to the 5′ end of the sequence  62  and noncomplementary to the sequence of the target RNA  61 . The reverse transcription reaction gives a nicking enzyme recognition sequence-containing cDNA  65 . Using a primer  74  having a sequence  72  having a sequence specific (complementary) to the cDNA  65  (namely a sequence specific (complementary) to the complementary sequence relative to the target RNA) and having a nicking enzyme recognition sequence-containing sequence  73 , and a primer  76  capable of hybridizing with the cDNA  65  on the side downstream from the primer  74  (on the side reverse to the direction of extension of the primer  74 ), the extension reaction is carried out with the cDNA  65  as a template. The primer  76  having a sequence  75  specific (complementary) to the cDNA  65  is extended while stripping off the primer  74 -derived extension product  81 . The primer  64  hybridizes with the extension product  81  stripped off and is extended to give a double-stranded DNA  83  having a plurality of nicking enzyme recognition sequences. Thereafter, a nicking enzyme is caused to act on the DNA  83  to give a nick-containing double-stranded DNA  84 . The DNA strand extension is effected under the action of a DNA polymerase having strand displacement activity while the nicks formed serve as priming sites, whereupon the nicking enzyme recognition sequence-containing double-stranded DNA  83  is regenerated. A nicking enzyme is again caused to act on the double-stranded DNA  83  to form the nick-containing DNA  84  and the DNA strand extension is carried out under the action of a DNA polymerase having strand displacement activity with the nicks serving as priming sites, whereupon the double-stranded DNA  83  is formed. By repeating these steps, it becomes possible to amplify a specific region of the target RNA under substantially isothermal conditions, namely at a temperature of 30° C.-75° C. 
         [0024]    As examples of the nicking enzyme which can be used in the practice of the invention, there may be mentioned BstNB (recognition sequence: GAGTC), N.AlwI (recognition sequence: GGATC), H. BbvCIA (recognition sequence: GCTGAGG), and N. BbvCIB (recognition sequence: CCTCAGC). As examples of the DNA polymerase having strand displacement activity which can be utilized in the practice of the invention, there may be mentioned Bst DNA polymerase, Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-) DNA polymerase, 9° Nm DNA polymerase, Terminator DNA polymerase, Klenow fragment of  E. coli  DNA polymerase I, Klenow fragment (3′→5′ exo-), and Phi29 DNA polymerase. 
         [0025]    The following specific embodiments illustrate the present invention in more detail. These embodiments are, however, by no means limitative of the scope of the invention. 
       First Embodiment 
     1. Primers Used in the First Embodiment 
       [0026]    Forward side primer for nicking enzyme recognition sequence introduction: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                 5′-gtggt gagtc acaac ggtgg ctgga cccca gga-3′ 
               
             
          
         
       
     
         [0027]    Forward side primer: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 5′-caagg gcctt tgcgt cag-3′ 
                 (SEQ ID NO; 2) 
               
             
          
         
       
     
         [0028]    Reverse side primer for nicking enzyme recognition sequence introduction: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 (SEQ ID NO: 3) 
               
             
          
           
               
                   
                 5′-gtggt gagtc acaac gcccc tgggc tcacc ccc-3′ 
               
             
          
         
       
     
         [0029]    Reverse side primer 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 5′-atctg ggaga caggc agg-3′ 
                 (SEQ ID NO: 4) 
               
             
          
         
       
     
       2. Composition of the Reaction Mixture Used in the First Embodiment 
     Each Value in the Parentheses being a Final Concentration 
       [0030]    Tris-HCl, pH 8.2 (15 mM), KCl (80 mM), (NH 4 ) 2 SO 4  (5 mM), MgSO 4  (1 mM), MgCl 2  (5 mM), DTT (0.5 mM), dATP (0.3 mM), dCTP (0.3 mM), dGTP (0.3 mM), dTTP (0.3 mM), Triton X-100 (0.05%) 
       3. Enzyme Composition Used in the First Embodiment 
       [0031]    Bst DNA Polymerase 8 U, N.BstNBI Nicking Enzyme 10 U 
         [0032]    For confirming whether target gene amplification is possible according to the flow chart illustrating the first aspect of the invention, amplified product detection was carried out by electrophoresis. The human genome was used as a template, and the primers listed above under 1 were used for amplifying the insulin gene. The primer  4  used was a nicking enzyme recognition sequence introducing forward primer in which bases 1-10 from the 5′ end constituted a nicking enzyme recognition sequence-containing sequence, bases 11-15 constituted a linker sequence, and bases 16-33 constituted a nicking enzyme recognition sequence introducing forward primer comprising a sequence specific to the insulin gene. The primer  6  was a forward primer comprising a sequence specific to the insulin gene. The primer  14  was a nicking enzyme recognition sequence introducing reverse primer in which bases 1-10 from the 5′ end constituted a nicking enzyme recognition sequence-containing sequence, bases 11-15 constituted a linker sequence, and bases 16-33 constituted a sequence specific to the insulin gene. The primer  16  was a reverse primer comprising a sequence specific to the insulin gene. 
         [0033]    The composition of the amplification reaction mixture and the enzyme composition were as shown above under 1 and 2, respectively. The reaction mixture containing the human genome sample and primers was set on a heat block set at 60° C. and incubated for 120 minutes. Thereafter, the reaction product obtained was electrophoresed on a chip electrophoretic apparatus (SV1210, product of Hitachi High-Technologies). The results of the electrophoresis are shown as a gel image  90  in  FIG. 5 . Lane  1  indicates the results of electrophoresis of size markers, while lane  2  shows the result of electrophoresis of the reaction product. As a result, the expected  112  bpamplified product  91  could be detected. This result indicates that the desired amplification product was obtained by the present invention in its first aspect. Thus, it was confirmed that genes can be isothermally amplified in accordance with the present invention. 
       Second Embodiment 
     1. Primers Used in the Second Embodiment 
       [0034]    Nicking enzyme recognition sequence introducing primer for reverse transcription: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 (SEQ ID NO: 5) 
               
             
          
           
               
                   
                 5′-catta gagtc tgttg tcgca agcac cctat cag-3′ 
               
             
          
         
       
     
         [0035]    Forward side primer for nicking enzyme recognition sequence introduction 
         [0000]    
       
         
               
             
               
               
             
           
               
                 (SEQ ID NO: 6) 
               
             
          
           
               
                   
                 5′-catta gagtc tgttg aatgc ctgga gattt ggg-3′ 
               
             
          
         
       
     
         [0036]    Forward side primer: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 5′-tttct tggat aaacc cgc-3′ 
                 (SEQ ID NO: 7) 
               
             
          
         
       
     
       2.Molecular Beacon Probe used in the Second Embodiment Molecular Beacon Probe for Detection:  
       [0037]      
         [0000]    
       
         
               
             
               
             
           
               
                 (SEQ ID NO: 8) 
               
             
          
           
               
                 5′-cgacg tggga aatcg cgtgt agtat gggac gtcg-3′ 
               
             
          
         
       
     
       3. Composition of the Reaction Mixture Used in the Second Embodiment 
     Each Value in the Parentheses being a Final Concentration 
       [0038]    Tris-HCl, pH 8.4 (35 mM), KCl (5 mM), NaCl (50 mM), (NH 4 ) 2 SO 4  (5 mM), MgSO 4  (1 mM), MgCl 2  (5 mM), DTT (0.5 mM), dATP (0.3 mM), dCTP (0.3 mM), dGTP (0.3 mM), dTTP (0.3 mM), Triton X-100 (0.05%) 
       4. Enzyme Composition Used in the First Embodiment 
       [0039]    Bst DNA Polymerase 16 U, N.BstNBI Nicking Enzyme 10 U 
         [0040]    For checking whether a target gene can be amplified using the flow according to the third aspect of the invention, amplified product detection was carried out using a real-time detection system. The template used was RNA extracted from hepatitis C virus (HCV), and the primers specified above under 1 were used for reverse transcription of the viral RNA and for amplification. The primer  64  used was a nicking enzyme recognition sequence introducing primer for reverse transcription in which bases 1-10 from the 5′ end constituted a nicking enzyme recognition sequence-containing sequence, bases 11-15 constituted a linker sequence, and bases 16-33 constituted a nicking enzyme recognition sequence introducing primer for reverse transcription comprising a sequence specific to the viral RNA. The primer  74  was a forward primer for nicking enzyme recognition sequence introduction in which bases 1-10 from the 5′ end constituted a nicking enzyme recognition sequence-containing sequence, bases 11-15 constituted a linker sequence, and bases 16-33 constituted a sequence specific to the cDNA obtained from the viral RNA by the reverse transcription reaction. The primer  76  was a forward primer comprising a sequence specific to the cDNA obtained from the viral RNA by the reverse transcription reaction. The detection probe used was the molecular beacon probe described above under 2. The molecular beacon probe was labeled with FAM at the 5′ end and with BHQ1 at the 3′ end and, in the probe, bases 1-6 and bases 29-34 from the 5′ end constituted stem sequences, and bases 7-28 constituted a sequence for hybridization with the amplified product. 
         [0041]    The composition of the reaction mixture and the specific examples of the enzyme composition were as shown above under 3 and 4, respectively. First, the reverse transcription reaction was carried out in the conventional manner using the revere transcription primers for cDNA production. Specifically, the reaction mixture containing the RNA sample to serve as a template and the reverse transcription primers was set on a heat block set at 42° C. and incubated for 60 minutes. Simultaneously, the reverse transcription reaction was carried out in the same manner as a negative control using a viral RNA-free sample. Thereafter, the reaction mixture containing the reverse transcription product, the reverse transcription primers and the two forward primers was set on a fluorescent microplate reader (Corona Electric) set at 63° C., and the time course of changes in reaction mixture fluorescent intensity was followed. For the negative control, the same measurement was carried out. The measurement results are shown in  FIG. 6  in the form of a graph  95 . The abscissa denotes the time, and the ordinate denotes the FAN fluorescent intensity. In the graph, the black dot plots show the amplification product-due fluorescent intensity data, and the white triangular plots show the viral RNA-free negative control-due fluorescent intensity data, and the respective results are shown in one and the same graph. The black dot plots show increases in FAN fluorescent intensity with time; thus, the amplification product formation in accordance with the flow chart illustrating the third aspect of the invention could be confirmed. On the other hand, the white triangular plots show no changes and thus it was found that no amplification product was formed. In view of the foregoing, the target RNA can be amplified and detected in accordance with the present invention. 
         [0042]    The invention is useful in amplifying nucleic acids (DNAs or RNAs) in gene analysis. It can also be utilized as a method of judging the presence or absence of specific prokaryotic or eukaryotic genes and as a method of detecting nucleic acids amplified by that method.