Circular probe amplification (cpa) using energy-transfer primers

Abstract

The present invention provides methods and kits for the rapid exponential amplification of nucleic acid molecules using a padlock probe. The present invention improves upon the existing methods for amplifying padlock probes by eliminating or delaying the appearance of artifact products that cause false positive results, and also increase the sensitivity and speed of the assay. Further provided are nucleic acid amplification primers containing non-informative base analogs.

What is claimed is:  
     
         1 . A nucleic acid amplification method comprising: 
 (a) providing a closed circular padlock probe molecule; a target nucleic acid molecule; a forward primer; a reverse primer; dNTPs; and a first DNA polymerase to form a reaction mixture;    (b) creating a multi-tailed complex;    (c) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and    (d) thermocycling said multi-tailed complex.    
     
     
         2 . The method of  claim 1 , wherein said first DNA polymerase is Bst LF.  
     
     
         3 . The method of  claim 1 , wherein said second DNA polymerase is a polymerase enzyme derived from  Thermus aquaticus , Pyrococcus species,  Thermococcus litoralis  or  Pyrococcus furiosus.    
     
     
         4 . The method of  claim 1 , wherein said second DNA polymerase is a recombinant thermostable polymerase.  
     
     
         5 . The method of  claim 4 , wherein the recombinant thermostable polymerase is an antibody-inactivated polymerase or a chemically-inactivated polymerase.  
     
     
         6 . The method of  claim 1 , wherein the reaction mixture further contains a strand displacement factor.  
     
     
         7 . The method of  claim 1 , wherein the reaction mixture is incubated for about 1 to about 30 minutes at about 50 to about 70° C. prior to thermocycling.  
     
     
         8 . The method of  claim 1 , wherein the reaction mixture is incubated for about 3 to about 20 minutes at about 50 to about 70° C. prior to thermocycling.  
     
     
         9 . The method of  claim 1 , wherein the method further comprises: 
 (e) denaturing said first DNA polymerase.    
     
     
         10 . The method of  claim 1 , wherein said target nucleic acid molecule is a single-stranded DNA molecule or a double-stranded DNA molecule.  
     
     
         11 . The method of  claim 1 , wherein said target nucleic acid molecule is a single-stranded RNA molecule or a double-stranded RNA molecule.  
     
     
         12 . The method of  claim 1 , wherein said target nucleic acid is a plasmid or fragment thereof, a genomic DNA or fragment thereof, a viral DNA or fragment thereof, a viral RNA or fragment thereof, an mRNA, a mitochondrial DNA or fragment thereof, or a chromosomal DNA or fragment thereof.  
     
     
         13 . The method of  claim 1 , wherein the 5′ terminal region of said forward or reverse primer is a tailed primer molecule.  
     
     
         14 . The method of  claim 1 , wherein at least one of the forward primer or reverse primer is a hairpin primer.  
     
     
         15 . The method of  claim 14 , wherein the hairpin primer has a loop region that contains a restriction endonuclease cleavage site.  
     
     
         16 . The method of  claim 1 , wherein at least one of said forward primer or reverse primer is detectably labeled.  
     
     
         17 . The method of  claim 16 , wherein the detectably labeled primer contains a molecular energy transfer mechanism.  
     
     
         18 . The method of  claim 1 , wherein at least one of said forward primer or reverse primer contains at least one non-informative base analog.  
     
     
         19 . The method of  claim 18 , wherein said non-informative base analog is nitropyrrole or nitroindole.  
     
     
         20 . The method of  claim 1 , wherein at least one of said forward primer or reverse primer contains at least one polarity switch.  
     
     
         21 . A nucleic acid molecule amplification kit comprising: 
 (a) a forward primer and a reverse primer;    (b) a ligase enzyme;    (c) a first polymerase enzyme;    (d) a linear padlock probe molecule, wherein the padlock probe comprises a 3′ terminal region, a 5′ terminal region, and a spacer region, wherein the spacer region contains binding sites for the forward primer and the reverse primer; and    (e) a second polymerase enzyme, wherein the second polymerase enzyme is a thermostable enzyme and wherein the second polymerase enzyme is not the same enzyme as the first polymerase enzyme.    
     
     
         22 . The kit of  claim 21 , wherein said first polymerase enzyme is Bst LF.  
     
     
         23 . The kit of  claim 21 , wherein said second polymerase is a polymerase enzyme derived from  Thermus aquaticus , Pyrococcus species,  Thermococcus litoralis  or  Pyrococcus furiosus.    
     
     
         24 . The kit of  claim 21 , wherein said second polymerase is a recombinant thermostable polymerase.  
     
     
         25 . The kit of  claim 24 , wherein the recombinant thermostable polymerase is an antibody-inactivated polymerase or a chemically-inactivated polymerase.  
     
     
         26 . The kit of  claim 21 , wherein at least one of the forward primer or reverse primer is a hairpin primer.  
     
     
         27 . The kit of  claim 26 , wherein the hairpin primer has a loop region that contains a restriction endonuclease cleavage site.  
     
     
         28 . The kit of  claim 21 , wherein at least one of said forward primer or reverse primer is detectably labeled.  
     
     
         29 . The kit of  claim 28 , wherein the detectably labeled primer contains a molecular energy transfer mechanism.  
     
     
         30 . The kit of  claim 21 , wherein at least one of said forward primer or reverse primer contains at least one non-informative base analog.  
     
     
         31 . The kit of  claim 30 , wherein said non-informative base analog is nitropyrrole or nitroindole.  
     
     
         32 . The kit of  claim 21 , wherein at least one of said forward primer or reverse primer contains at least one polarity switch.  
     
     
         33 . A method for detecting a target nucleic acid molecule in a sample comprising: 
 (a) providing a target nucleic acid molecule, a linear padlock probe molecule, a ligase enzyme, a forward primer, a reverse primer, dNTPs, and a first DNA polymerase;    (b) creating a closed circular padlock probe molecule;    (c) creating a multi-tailed complex from said closed circular padlock probe molecule;    (d) activating a second DNA polymerase;    (e) thermocycling said multi-tailed complex with said second DNA polymerase; and    (f) detecting the amplification product of said multi-tailed complex.    
     
     
         34 . A method for detecting a target nucleic acid molecule in a sample comprising: 
 (a) providing a target nucleic acid molecule, a closed circular padlock probe molecule topologically linked to said target nucleic acid molecule, a forward primer, a reverse primer, dNTPs, and a first DNA polymerase;    (b) creating a multi-tailed complex from said closed circular padlock probe molecule;    (c) activating a second DNA polymerase;    (d) thermocycling said multi-tailed complex with said second DNA polymerase; and    (e) detecting the amplification product of said multi-tailed complex.    
     
     
         35 . A method for detecting a plurality of target nucleic acid molecules in a sample comprising: 
 (a) providing a plurality of target nucleic acid molecules, a plurality of linear padlock probe molecules capable of annealing to a plurality of distinct target nucleic acid molecule, a ligase enzyme, dNTPs, and a first DNA polymerase;    (b) creating at least two closed circular nucleic acid molecules, wherein each of said closed circular nucleic acid molecules is topologically linked to a distinct target nucleic acid molecule;    (c) providing, for each member of said at least two closed circular nucleic acid molecules, a forward primer and a reverse primer;    (d) creating a multi-tailed complex for each of said distinct target nucleic acid molecules;    (e) activating a second DNA polymerase;    (f) thermocycling said at least two distinct multi-tailed complexes with said second DNA polymerase; and    (g) detecting the amplification products of said at least two distinct multi-tailed complexes.    
     
     
         36 . The method of  claim 35 , wherein said detection of the amplification products of the multi-tailed complexes is performed by a real-time detection instrument.  
     
     
         37 . The method of  claim 35 , wherein said first DNA polymerase is Bst LF.  
     
     
         38 . The method of  claim 35 , wherein said second DNA polymerase is a polymerase enzyme derived from  Thermus aquaticus , Pyrococcus species,  Thermococcus litoralis  or  Pyrococcus furiosus.    
     
     
         39 . The method of  claim 35 , wherein said second DNA polymerase is a recombinant thermostable polymerase.  
     
     
         40 . The method of  claim 39 , wherein the recombinant thermostable polymerase is an antibody-inactivated polymerase or a chemically-inactivated polymerase.  
     
     
         41 . The method of  claim 35 , wherein the reaction mixture further contains a strand displacement factor.  
     
     
         42 . The method of  claim 35 , wherein the reaction mixture is incubated for about 1 to about 30 minutes at about 50 to about 70° C. prior to thermocycling.  
     
     
         43 . The method of  claim 35 , wherein the reaction mixture is incubated for about 3 to about 20 minutes at about 50 to about 70° C. prior to thermocycling.  
     
     
         44 . The method of  claim 35 , wherein the method further comprises: 
 (e) denaturing said first DNA polymerase.    
     
     
         45 . The method of  claim 35 , wherein, for at least one of said at least two closed circular nucleic acid molecules, the 5′ terminal region of said forward primer or reverse primer does not hybridize to the closed circular nucleic acid molecule.  
     
     
         46 . The method of  claim 35 , wherein, for at least one of said at least two closed circular nucleic acid molecules, at least one of said forward primer or reverse primer is a hairpin primer.  
     
     
         47 . The method of  claim 46 , wherein the hairpin primer has a loop region that contains a restriction endonuclease cleavage site.  
     
     
         48 . The method of  claim 35 , wherein, for at least one of said at least two closed circular nucleic acid molecules, at least one forward or reverse primer is labeled with a detectable label.  
     
     
         49 . The method of  claim 48 , wherein the detectable label is a distinguishable detectable label.  
     
     
         50 . The method of  claim 35 , wherein, for at least one of said at least two closed circular nucleic acid molecules, at least one of said forward primer or reverse primer contains at least one non-informative base analog.  
     
     
         51 . The method of  claim 50 , wherein said modified non-informative base analog is nitropyrrole or nitroindole.  
     
     
         52 . The method of  claim 35 , wherein, for at least one of said at least two closed circular nucleic acid molecules, at least one of said forward primer or reverse primer contains at least one polarity switch.  
     
     
         53 . A closed tube nucleic acid molecule amplification method comprising: 
 (a) providing a target nucleic acid molecule, a ligase enzyme; a forward primer; a reverse primer; dNTPs; and a first DNA polymerase in a reaction tube;    (b) sealing said reaction tube;    (c) creating a closed circular padlock probe molecule;    (d) creating a multi-tailed complex from said closed circular padlock probe molecule;    (e) activating a second DNA polymerase, wherein said second DNA polymerase is a thermostable DNA polymerase; and    (f) thermocycling said multi-tailed complex.    
     
     
         54 . The method of  claim 53 , wherein the ligase enzyme is a thermolabile ligase enzyme.  
     
     
         55 . The method of  claim 53 , wherein at least one dNTP is a caged dNTP.  
     
     
         56 . A padlock probe amplification primer comprising any of the following sequences:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-actagagctgagaca-3′; 
                     
                 
                     
                     
                 
                     
                   5′-actagagttcagaca-3′; 
                 
                     
                     
                 
                     
                   5′-actagagctgagacatgacga-3′; 
                 
                     
                     
                 
                     
                   5′-actagagucagacatgacga-3′; 
                 
                     
                     
                 
                     
                   5′-actagagctgagacatgacgagtc-3′; 
                 
                     
                     
                 
                     
                   5′-actagagttcagacatgacgagtc-3′; 
                 
                     
                     
                 
                     
                   5′-actagagctgagacatgacgagtcgca-3′; or 
                 
                     
                     
                 
                     
                   5′-actagagttcagacatgacgagtcgca-3′ 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein at least one nucleotide base contained in the primer is a non-informative base analog.  
       
     
     
         57 . The padlock probe amplification primer of  claim 56 , wherein the non-informative base analog is nitropyrrole or nitroindole.  
     
     
         58 . The padlock probe amplification primer of  claim 56 , wherein the non-informative base analog is a polarity switch.  
     
     
         59 . A padlock probe amplification primer of 15 to 75 nucleotides wherein at least one nucleotide base contained in the primer is a non-informative base analog.  
     
     
         60 . The padlock probe amplification primer of  claim 59 , wherein the non-informative base analog is nitropyrrole or nitroindole.  
     
     
         61 . The padlock probe amplification primer of  claim 59 , wherein the non-informative base analog is a polarity switch.