Method for the non-specific amplification of nucleic acid

Abstract

The present invention is concerned with a method for generating, in a non specific manner, multiple copies of RNA from a pool of mRNA's. Such a method is of particular importance in techniques for screening the differences in expression in given cell types or in cells under specific conditions. The present invention provides a non-selective poly A mRNA labeling and amplification method, i.e. a method not encompassing cDNA synthesis. The present invention is directed to a method for amplifying RNA by creating, in a non specific manner, multiple RNA copies starting from nucleic acid containing starting material comprising a pool of mRNA's each mRNA comprising a poly-A tail, wherein the material is contacted simultaneously with an oligonucleotide comprising an oligo-dT sequence, the sequence of a promoter recognized by an RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter, and further with an enzyme having reverse transcriptase activity, an enzyme having RNase H activity and an enzyme having RNA polymerase activity and the necessary nucleotides and the resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the enzymatic processes to take place. In a preferred method of the invention the generated RNA copies made are contacted with an RNA ligase, a double stranded nucleic acid complex comprising a double stranded DNA promoter sequence that can be recognize by an RNA polymerase, whereby one strand of said complex has a stretch of RNA attached to the 5′ end of one of the DNA strands, an enzyme having RNA polymerase activity, and the necessary nucleotides. The resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the amplification to take place.

1 . Method for creating, in a non specific manner, multiple RNA copies starting from nucleic acid containing starting material comprising a pool of mRNA's each mRNA comprising a poly-A tail wherein the material is simultaneously contacted with: 
 an oligonucleotide comprising an oligo-dT sequence, the sequence of a promoter recognized by a RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter,    an enzyme having reverse transcriptase activity    an enzyme having RNase H activity and    an enzyme having RNA polymerase activity,    the necessary nucleotides    and the resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the enzymatic processes to take place.    
     
     
         2 . Method according to  claim 1 , wherein the oligonucleotide is blocked at its 3′ end.  
     
     
         3 . Method according to  claim 1  or  2 , wherein the promoter sequence is the T7-promoter sequence and the RNA polymerase is T7 RNA polymerase.  
     
     
         4 . Method according to any of the preceding claims wherein the enzyme having reverse transcriptase activity is AMV-RT or MMLV-RT.  
     
     
         5 . Method according to any of the preceding claims wherein (one of) the enzyme(s) having RNase H activity is  E. coli  RNase H.  
     
     
         6 . Method according to  claim 1  wherein the enzyme having RNase H activity is a reverse transcriptase.  
     
     
         7 . Method according to  claim 6  wherein the enzyme having RNase H activity is AMV-RT or MMLV-RT.  
     
     
         8 . Method according to any of the preceding claims, wherein at least one of the nucleotides is provided with a label.  
     
     
         9 . Method according to any of the preceding claims wherein the generated RNA is used as input material for further amplification.  
     
     
         10 . Method according to  claim 9  wherein the generated RNA copies made by the method described in  claim 1  are contacted with 
 a RNA ligase,  
 a double stranded nucleic acid complex comprising a double stranded DNA promoter sequence that can be recognized by a RNA polymerase, whereby one strand of said complex has a stretch of RNA attached to the 5′ end of one of the DNA strands,  
 an enzyme having RNA polymerase activity,  
 the necessary nucleotides  
 and the resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the enzymatic processes amplification to take place  
 
     
     
         11 . Method according to  claim 10 , wherein the stretch of RNA attached to the 5′ end of one of the DNA strands is phosphorylated at the 5′ end  
     
     
         12 . Method according to  claim 10  or  11 , wherein the promoter sequence is the T7-promoter sequence and the RNA polymerase is T7 RNA polymerase.  
     
     
         13 . Method according to  claim 10 , wherein at least one of the nucleotides is provided with a label.  
     
     
         14 . Method according to  claim 1 , wherein the reaction mixture further comprises an RNA ligase and a double stranded nucleic acid complex comprising a double stranded DNA promoter sequence that can be recognized by the RNA polymerase, whereby one strand of said complex has a stretch of RNA attached to the 5′ end of one of the DNA strands.  
     
     
         15 . Method according to  claim 1 , wherein the generated RNA copies made by the method described in  claim 1  are contacted with 
 a poly A polymerase.