Random-primed reverse transcriptase - in vitro transcription method for rna amplification

Abstract

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

1 . A kit for use in amplifying one or more single stranded nucleic acids, said kit comprising in one or more containers: 
 (i) a first set of oligonucleotides, each of said oligonucleotides in said first set comprising a promoter sequence operably linked to a random sequence at least 4 nucleotides in length from a set of random sequences of at least 4 nucleotides; and    (ii) a second set of oligonucleotides, each of said oligonucleotides in said second set comprising a random sequence of at least 4 nucleotides in length from a set of random sequences of at least 4 nucleotides.    
     
     
         2 . The kit of  claim 1 , which further comprises a reverse transcriptase.  
     
     
         3 . The kit of  claim 1 , which further comprises an RNA polymerase that recognizes said promoter sequence.  
     
     
         4 . The kit of  claim 1 , wherein the random sequences of the oligonucleotides in said first set of oligonucleotides are 6 to 9 nucleotides in length.  
     
     
         5 . The kit of  claim 1 , wherein the random sequences of the oligonucleotides in said second set of oligonucleotides are 6 to 9 nucleotides in length.  
     
     
         6 . The kit of  claim 1 , wherein the random sequences of the oligonucleotides in said first set of oligonucleotides are 6 nucleotides in length.  
     
     
         7 . The kit of  claim 1 , wherein the random sequences of the oligonucleotides in said second set of oligonucleotides are 6 nucleotides in length.  
     
     
         8 . The kit of  claim 1 , wherein the oligonucleotides in said second set of oligonucleotides do not comprise a promoter sequence.  
     
     
         9 . The kit of  claim 1 , wherein each oligonucleotide in said second set of oligonucleotides consists of a random sequence of at least 4 nucleotides in length from the set of random sequences of at least 4 nucleotides.  
     
     
         10 . The kit of  claim 1 , which further comprises a third set of oligonucleotides, each of said oligonucleotides in said third set comprising the promoter sequence operably linked to a polydT sequence of at least 5 nucleotides.  
     
     
         11 . The kit of  claim 10 , wherein said polydT sequence is 5 to 25 nucleotides.  
     
     
         12 . The kit of  claim 11 , wherein said polydT sequence is 18 nucleotides.  
     
     
         13 . The kit of  claim 3 , wherein said promoter sequence is a T7 promoter sequence, and said RNA polymerase is T7 RNA polymerase.  
     
     
         14 . The kit of  claim 1 , which further comprises a reverse transcriptase inhibitor.  
     
     
         15 . The kit of  claim 14 , wherein the reverse transcriptase inhibitor comprises ddNTP.