Isothermal dna amplification

Abstract

The present invention provides for amplification of one or more polynucleotides by multi-staged linear amplifications using one or more RNA polymerases. At each stage RNA transcripts are accumulated at a linear rate, so that multiple stages provide for faster than linear transcript accumulation. In one aspect, the invention provides for polynucleotide amplification by ligating hairpin adaptors to an end of polynucleotides wherein the hairpin adaptors each contain a promoter sequence oriented so that transcription proceeds in the direction of the loop of the hairpin adaptor. Upon transcription through such loop region and to the complementary strand a replicate is made of the promoter sequence as well as the polynucleotide, thereby permitting exponential amplification upon reverse transcription, second strand synthesis, and repetition of the above cycle. Preferably such amplification is carried out under isothermal reaction conditions.

1 - 16 . (canceled) 
     
     
         17 . A polynucleotide comprising:
 a polynucleotide sequence;   an adapter at one end of the polynucleotide sequence comprising a hairpin; and   a promoter sequence positioned between the polynucleotide sequence and the hairpin, wherein the promoter sequence is oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin.   
     
     
         18 . The polynucleotide of  claim 17 , wherein the adapter comprises the promoter sequence. 
     
     
         19 . The polynucleotide of  claim 17 , wherein the hairpin contains a loop region of from three to six nucleotides. 
     
     
         20 . The polynucleotide of  claim 17 , wherein the polynucleotide further comprises a primer binding site at the end opposite to that of the hairpin adapter. 
     
     
         21 . The polynucleotide of  claim 20 , wherein the primer binding site is present in a second adapter. 
     
     
         22 . The polynucleotide of  claim 17 , wherein the RNA polymerase is selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase. 
     
     
         23 . The polynucleotide of  claim 17 , wherein the polynucleotide sequence is double stranded. 
     
     
         24 . The polynucleotide of  claim 17 , wherein the polynucleotide sequence is single stranded. 
     
     
         25 . The polynucleotide of  claim 17 , wherein the polynucleotide is attached to a solid phase support on the end opposite to that of the hairpin adapter. 
     
     
         26 . The polynucleotide of  claim 25 , wherein the polynucleotide is attached to the solid phase support through its 5′ end. 
     
     
         27 . The polynucleotide of  claim 25 , wherein the polynucleotide is covalently attached to the solid phase support. 
     
     
         28 . The polynucleotide of  claim 25 , wherein the polynucleotide is non-covalently attached to the solid phase support. 
     
     
         29 . A hairpin adapter comprising a loop region and a double stranded region, wherein the double stranded region comprises a promoter sequence oriented so that synthesis by an RNA polymerase recognizing the promoter sequence proceeds in the direction of the hairpin. 
     
     
         30 . The hairpin adapter of  claim 17 , wherein the hairpin contains a loop region of from three to six nucleotides. 
     
     
         31 . A kit comprising the hairpin adapter of  claim 29 . 
     
     
         32 . The kit of  claim 31 , further comprising:
 reagents for attachment of the hairpin adapter to a polynucleotide of interest; and   reagents for amplification of a polynucleotide having the adapter attached thereto.   
     
     
         33 . The kit according to  claim 32 , wherein the reagents for attachment and amplification comprise: a ligase, an RNA polymerase recognizing the promoter sequence, a reverse transcriptase, and a DNA polymerase. 
     
     
         34 . The kit according to  claim 32 , further comprising a solid phase support and reagents for attaching a polynucleotide of interest thereto.