Nucleic acid promoters

Abstract

Disclosed is a method of detecting the presence of a nucleic acid target sequence of interest, the method comprising the steps of: (a) adding first and second nucleic acid probes to a sample comprising the sequence of interest, so as to form a complex comprising three strands of nucleic acid, wherein the first probe comprises the full length sequence of a first strand of a double stranded promoter, the target sequence comprises an end part of a second strand of the double stranded promoter which is complementary to a part of the first strand, and the second probe comprises the rest of the second strand of the double stranded promoter which is complementary to a part of the first strand, such that a functional promoter is formed when the first probe is hybridised to both the target sequence and to the second probe; (b) adding a polymerase which recognises the promoter, so as to cause the de novo synthesis of nucleic acid from the promoter present in the complex; and (c) detecting directly or indirectly the de novo synthesised nucleic acid. Also disclosed is the complex formed in performance of the method defined above, and a kit for performing the method defined above.

1 . A method of detecting the presence of a nucleic acid target sequence of interest, the method comprising the steps of: 
 (a) adding first and second nucleic acid probes to a sample comprising the sequence of interest, so as to form a complex comprising three strands of nucleic acid, wherein the first probe comprises the full length sequence of a first strand of a double stranded promoter, the target sequence comprises an end part of a second strand of the double stranded promoter which is complementary to a part of the first strand, and the second probe comprises the rest of the second strand of the double stranded promoter which is complementary to a part of the first strand, such that a functional promoter is formed when the first probe is hybridised to both the target sequence and to the second probe;    (b) adding a polymerase which recognises the promoter, so as to cause the de novo synthesis of nucleic acid from the promoter present in the complex; and    (c) detecting directly or indirectly the de novo synthesised nucleic acid:    
     
     
         2 . A method according to  claim 1 , wherein the promoter is an RNA polymerase promoter and the de novo synthesised nucleic acid is RNA.  
     
     
         3 . A method according to  claim 1  or  2 , wherein the promoter is recognised by T3, T7or SP6 RNA polymerase or a mutant form thereof.  
     
     
         4 . A method according to any one of the preceding claims, wherein the second probe comprises a template portion which may act as a template for synthesis of nucleic acid from the functional promoter.  
     
     
         5 . A method according to  claim 4 , wherein the second probe comprises a +12 region adjacent to the promoter to optimise transcription from the promoter.  
     
     
         6 . A method according to  claim 5 , wherein the second probe comprises a +12 region sequence selected from the group consisting of:  
       
         
           
                 
                 
               
                     
                 
                   (5′® 3′)   GTTCTCTCTCCC;    GCTCTCTCTCCC;    GTTGTGTCTCCC; 
                     
                 
                     
                 
                      GATGTGTCTCCC; ATCCTCTCTCCC; GTTCTCGTGCCC; ATCCTCGTGCCC; 
                 
                     
                 
                      GCTCTCGTGCCC; and GTTGTGGTGCCC. 
                 
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . A method according to any one of claims  4 ,  5  or  6 , wherein the template portion, when copied by the polymerase, provides a sequence which can act as an RNA polymerase promoter, or may be used for detection and/or capture at a solid surface.  
     
     
         8 . A method according to any one of claims  4 - 7 , wherein the template portion, when copied by the polymerase, provides a sequence which hybridises with a molecular beacon.  
     
     
         9 . A method according to any one of claims  4 - 7 , wherein the template portion, when copied by the polymerase, provides a sequence which acts as a ribozyme.  
     
     
         10 . A method according to any one of the preceding claims, wherein the de novo synthesised nucleic acid is subjected to an amplification step prior to detection.  
     
     
         11 . A method according to  claim 10 , wherein the amplification step comprises: hybridising the de novo synthesised nucleic acid to a third nucleic acid probe, which hybridisation forms a second double stranded nucleic acid promoter either directly, or by 3′ extension of the de novo synthesised nucleic acid using the third probe as template; and adding a polymerase which recognises the second promoter so as to cause nucleic acid synthesis therefrom.  
     
     
         12 . A method according to  claim 10 , wherein the nucleic acid synthesised from the second promoter is detected.  
     
     
         13 . A method according to  claim 11 , further comprising the steps of: hybridising the nucleic acid synthesised from the second promoter to a fourth nucleic acid probe, which hybridisation forms a third double stranded nucleic acid promoter either directly, or by 3′ extension of the nucleic acid synthesised from the second promoter using the fourth probe as template; and adding a polymerase which recognises the third promoter so as to cause nucleic acid synthesis therefrom.  
     
     
         14 . A method according to  claim 13 , wherein nucleic acid synthesised from the third promoter is detected.  
     
     
         15 . A method according to  claim 13 , further comprising the step of hybridising nucleic acid synthesised from the third promoter to the third probe, thereby reforming the second double stranded promoter, so as to create a cycle of nucleic acid synthesis.  
     
     
         16 . A method according to  claim 10 , wherein the amplification step comprises: adding third and fourth nucleic acid probes so as to form a complex comprising the said probes and the de novo synthesised nucleic acid, wherein the third probe comprises the full length sequence of a first strand of a double stranded promoter, the de novo synthesised nucleic acid comprises an end part of a second strand of the double stranded promoter which is complementary to a part of the first strand, and the fourth probe comprises the rest of the second strand of the double stranded promoter which is complementary to a part of the first strand, such that a functional promoter is formed when the third probe is hybridised to both the de novo synthesised nucleic acid and to the fourth probe; adding a polymerase which recognises the promoter, so as to cause the synthesis of nucleic acid from the promoter present in the complex; and detecting directly or indirectly the synthesised nucleic acid.  
     
     
         17 . A nucleic acid complex comprising three strands of nucleic acid: a promoter strand, a promoter complementary strand, and a target strand; wherein the promoter complementary strand comprises the full length sequence of a first strand of a double stranded promoter; the target strand comprises a part of a second strand of the double stranded promoter which is complementary to a part of the first strand; and the promoter strand comprises a part of the second strand of the double stranded promoter which is complementary to a part of the first strand; wherein neither part of the second strand of the double stranded promoter present on the target strand or on the promoter strand is capable of forming a functional promoter when hybridised to the promoter complementary strand in the absence of the other part, but wherein a functional promoter is formed when the promoter complementary strand is hybridised to both the target strand and the promoter strand.  
     
     
         18 . A complex according to  claim 17 , wherein the promoter complementary strand and promoter strand are provided by first and second nucleic acid probes respectively, the complex being formed in performance of a method according to-any one of claims  1 - 16 .  
     
     
         19 . A kit for performing the method of  claim 1 , the kit comprising first and second probes for forming, together with the appropriate target sequence, the complex of  claim 17 , and instructions for performing the method of any one of claims  1 - 16 .  
     
     
         20 . A kit according to  claim 19 , further comprising one or more of the following: DNA polymerase; RNA polymerase; ribo- or deoxyribonucleotide triphosphates; labelling reagents; detection reagents; buffers.  
     
     
         21 . A method substantially as hereinbefore described and with reference to the accompanying drawings.