Nucleic acid amplication utilizing intermediate duplexes

Abstract

This invention provides for a novel amplification procedure for nucleic acid. The method uses a wild type or mutant RNA polymerase designed to transcribe both deoxyribonucleotides and ribonucleotides (see FIGS. 1 - 7 ).

1 . An oligonucleotide primer comprising in the following order from 5′ to 3′: 
 a phage-encoded RNA polymerase recognition sequence,    a spacer sequence comprising a sequence of from 12 to 21 nucleotides, and    a target complementary sequence which can bind a segment of a target nucleic acid.    
     
     
         2 . The primer of  claim 1 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XY) n , 
 wherein n is from 6 to 10,    wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide,    wherein X and Y are not the same.    
     
     
         3 . The primer of  claim 2 , wherein X is an adenine nucleotide and Y is a guanine nucleotide.  
     
     
         4 . The primer of  claim 3 , wherein n is 9.  
     
     
         5 . The primer of  claim 1 , wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides.  
     
     
         6 . The primer of  claim 5 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ) n , wherein n is from 4 to 7, 
 and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide.    
     
     
         7 . The primer of  claim 1 , wherein said spacer sequence comprises a nucleotide sequence having the formula (X) n , 
 wherein n is from 12 to 20,    wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide.    
     
     
         8 . The primer of  claim 7 , wherein n is 18.  
     
     
         9 . A method of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising: 
 i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end, 
 wherein said intermediate duplex comprises a double-stranded molecule, wherein said double-stranded DNA molecule comprises a first and a second strand,  
 wherein said first strand comprises in the following order from 5′ to 3: 
 a phage-encoded RNA polymerase recognition sequence,  
 a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, and  
 a first target complementary sequence which can bind to a segment of said target nucleic acid,  
 
 wherein said second strand comprises in the following order from 5′ to 3′: 
 a second target complementary sequence which can bind to a segment of said target nucleic acid,  
 a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and  
 a phage-encoded RNA polymerase recognition sequence,  
 
 wherein said transcribing takes place in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said first spacer sequence;  
   ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex, 
 wherein said second primer comprises in the following order from 5′ to 3′: 
 a phage-encoded RNA polymerase recognition sequence, said second spacer sequence, and  
 said second target complementary sequence which can bind to a 3′ segment of said target nucleic acid;  
 
   iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex;    iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said second spacer sequence, to form an antisense transcription product;    v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex, 
 wherein said first primer comprises in the following order from 5′ to 3′: 
 a phage-encoded RNA polymerase recognition sequence, said first spacer sequence, and  
 said first target complementary sequence which can bind to a 5′ segment of said target nucleic acid;  
 
   vi.) extending said second primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and    vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said first spacer sequence to form said sense transcription product.    
     
     
         10 . The method of  claim 9 , wherein the method further comprises repetitively carrying out steps i to vii.  
     
     
         11 . The method of  claim 9 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (XY) n , 
 wherein n is from 6 to 10,    wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thyrmidine nucleotide,    wherein X and Y are not the same.    
     
     
         12 . The method of  claim 11 , wherein X is an adenine nucleotide and Y is a guanine nucleotide.  
     
     
         13 . The method of  claim 12 , wherein n is 9.  
     
     
         14 . The method of  claim 9 , wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides.  
     
     
         15 . The method of  claim 14 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ) n , wherein n is from 4 to 7, 
 and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide.    
     
     
         16 . The method of  claim 9 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (X) n , 
 wherein n is from 12 to 20,    wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide.    
     
     
         17 . The method of  claim 16 , wherein n is 18.  
     
     
         18 . The method of  claim 9 , wherein said sense and antisense transcription products comprise a nucleic acid strand comprising both ribonucleotides and deoxyribonucleotides.  
     
     
         19 . The method of  claim 9 , wherein said first and said second amplification duplexes consist of deoxyribonucleotides and ribonucleotides.  
     
     
         20 . The method of  claim 9 , wherein said method is carried out at a single temperature.  
     
     
         21 . The method of  claim 9 , wherein said method is carried out at a single temperature of between 25° C. and 55° C.  
     
     
         22 . The method of  claim 9 , wherein the method is carried out at a single temperature of greater than 50° C.  
     
     
         23 . The method of  claim 9 , wherein said intermediate duplex comprises a double-stranded DNA comprising one complete primer sequence followed by the entire sequence that is to amplified.  
     
     
         24 . The method of  claim 9 , wherein said intermediate duplex is formed from double-stranded DNA, single-stranded DNA, or RNA.  
     
     
         25 . The method of  claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of: 
 denaturing a double-stranded DNA target to form an upper strand and a lower strand;    hybridizing said first primer to said lower strand to form a first primer-lower strand complex;    extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex;    denaturing said first long sense strand product-lower strand complex into a first long sense strand product and said lower strand;    hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and    extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.    
     
     
         26 . The method of  claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of: 
 denaturing a double-stranded DNA target to form an upper strand and a lower strand;    hybridizing said first primer to said lower strand to form a first primer-lower strand complex;    extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex, wherein said first long sense strand product has a 5′ and a 3′ end;    displacing said first sense strand product from said lower strand by: 
 hybridizing a bumper oligonucleotide to a subsequence on said lower strand adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;  
 extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first sense strand product; hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and  
   extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.    
     
     
         27 . The method of  claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of: 
 hybridizing said second primer to a target RNA molecule to form a second primer-RNA template complex;    extending said second primer-target RNA molecule complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex, wherein said first long antisense strand product has a 5′ and a 3′ end;    displacing said first long antisense strand product from said target RNA molecule by: 
 hybridizing a bumper oligonucleotide to a subsequence on said target RNA molecule adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;  
 extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first long antisense strand product;  
   hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and    extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.    
     
     
         28 . The method of  claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of: 
 hybridizing said second primer to a single-stranded target RNA molecule to form a second primer-RNA template complex;    extending said second primer-RNA template complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex;    denaturing said first long antisense strand product-RNA template complex into a first long antisense strand product and said single-stranded RNA molecule;    hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and    extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.    
     
     
         29 . The method of  claim 9 , wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase.  
     
     
         30 . The method of  claim 29 , wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid.  
     
     
         31 . The method of  claim 30 , wherein said phage-encoded RNA polymerase is a T7 RNA polymerase.  
     
     
         32 . The method of  claim 31 , wherein said T7 RNA polymerase contains a Y639F mutation.  
     
     
         33 . The method of  claim 31 , wherein said T7 RNA polymerase contains a S641A mutation.  
     
     
         34 . The method of  claim 31 , wherein said T7 RNA polymerase contains at least two mutations.  
     
     
         35 . The method of  claim 9 , wherein said Mn ++  is present in a concentration of between 10 μM to 20 mM.  
     
     
         36 . The method of  claim 35 , wherein said concentration is 10 mM.  
     
     
         37 . The method of  claim 9 , wherein said target nucleic acid is single-stranded DNA.  
     
     
         38 . The method of  claim 9 , wherein the target nucleic acid is comprised of RNA.  
     
     
         39 . The method of  claim 9 , further detecting said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex, 
 wherein said detecting comprises hybridizing a detection oligonucleotide comprising a detectable moiety, wherein said detection oligonucleotide is complementary to a subsequence of said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex.    
     
     
         40 . A kit for copying a target nucleic acid comprising: 
 a container containing:    a first nucleotide primer comprising in the following order from 5′ to 3′: 
 a phage-encoded RNA polymerase recognition sequence,  
 a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, 
 a first target complementary sequence which can bind to a segment of said target nucleic acid; and  
 
   a second primer comprising in the following order from 5′ to 3′: 
 a phage-encoded RNA polymerase recognition sequence,  
 a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and  
 a second target complementary sequence which can bind to a segment of said target nucleic acid.  
   
     
     
         41 . The kit of  claim 40 , wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase.  
     
     
         42 . The kit of  claim 41 , wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid.  
     
     
         43 . The kit of  claim 41 , wherein said phage-encoded RNA polymerase is a T7 RNA polymerase.  
     
     
         44 . The kit of  claim 43 , wherein said T7 RNA polymerase contains a Y639F mutation.  
     
     
         45 . The kit of  claim 43 , wherein said T7 RNA polymerase contains a S641 A mutation.  
     
     
         46 . The kit of  claim 43 , wherein said T7 RNA polymerase contains at least two mutations.  
     
     
         47 . The kit of  claim 40 , further comprising a member selected from the group consisting of: 
 a DNA polymerase;    a Reverse Transcriptase that lacks RNAseH activity;    a phage-encoded RNA polymerase;    all four dNTPs;    those rNTPs represented in said first and second spacer sequences;    reaction buffer containing manganese in a concentration from 10 μM to 20 mM and magnesium in a concentration from 5 mM to 20 mM;    a positive control target nucleic acid; and    instructions for carrying out a method of copying a nucleic acid using said first primer and said second primer.