Ligation-Based Rna Amplification

Abstract

Methods of amplification, purification and detection of nucleic acid sequences especially RNA are described. One aspect of the method involves the hybridisation and subsequent ligation of a nucleic acid structure to the nucleic acid sequence desired to be manipulated. The methods require that the nucleic acid structure comprises a double stranded region and a single stranded region. The single stranded region is complementary to the RNA sequence of interest. The double stranded region may also contain additional functionalities which are then used subsequently in the method.

1 . A method of producing a ligated nucleic acid molecule comprising: 
 a) supplying RNA other than poly A;    b) supplying one or more nucleic acids having a double stranded region and a single stranded 3′ terminal region; and    c) hybridizing the single stranded 3′ terminal region of the nucleic acid sequence to the RNA and ligating one 5′ end of the double stranded region of the nucleic acid to the 3′ end of the RNA by enzymatic means.    
     
     
         2 . The method of  claim 1 , wherein the nucleic acids of step b) comprise DNA.  
     
     
         3 . The method of  claim 1 , wherein the nucleic acids incorporates one or more features selected from the group consisting of: 
 a) a nucleotide sequence that can subsequently be used as a promoter sequence for RNA synthesis; and    b) a Tag which can be used to label the nucleic acid or manipulate the nucleic acid.    
     
     
         4 . The method of  claim 3 , further comprising transcribing the product obtained using the nucleotide sequence of 1) and of tag 2) with RNA polymerase to produce a 5′ sequence tagged cRNA.  
     
     
         5 . The method of  claim 4 , wherein the 5′ tagged cRNA molecule is ligated with a second double stranded DNA sequence comprising a double stranded region and a single stranded region.  
     
     
         6 . The method of  claim 5 , wherein the ligated RNA-DNA molecule product is further transcribed by RNA polymerase to produce multiple copies of RNA containing tags at the 5′ and 3′ end of the RNA molecule.  
     
     
         7 . The method of  claim 6 , wherein the 5′ and 3′ tagged RNA sequence is: 
 a) mixed and hybridized with a DNA primer which is complementary to the sequence tag at the 3′ end of the RNA;    b) incubated with reverse transcriptase and dNTPs to produce a single strand cDNA-RNA heteroduplex;    c) incubating the product of step b) with RNase; and    d) incubating the product of step c) with a second single strand primer the sequence of which is complementary to the tag sequence at the 3′ end of the single strand cDNA and DNA polymerase to produce a double stranded cDNA containing sequence tags at both ends.    
     
     
         8 . A method of amplifying a target RNA sequence comprising the steps of: 
 a) supplying the RNA in single stranded form;    b) adding a DNA sequence that comprises a double stranded region which contains a promoter sequence for RNA polymerase and a single stranded region which hybridizes to the target RNA;    c) ligating the DNA sequence to the 3′ end of RNA by enzymatic means to produce a DNA-RNA; and    d) transcribing the DNA-RNA with RNA polymerase to produce antisense complementary RNA (cRNA).    
     
     
         9 . The method of amplification of  claim 8 , further comprising: 
 e) adding a DNA sequence that comprises a double stranded region which contains a promoter sequence for RNA polymerase and a single stranded region which hybridizes to the cRNA;    f) ligating the cRNA and DNA sequence by enzymatic means; and    g) transcribing the product of step f) with RNA polymerase to produce multiple copies of RNA having the same sense as the target RNA.    
     
     
         10 . The method of amplification of  claim 9 , wherein at least one of the double stranded DNA sequence contains a sequence Tag.  
     
     
         11 . The method of amplification of  claim 9 , wherein both double stranded DNA sequence contain a sequence Tag.  
     
     
         12 . The method of amplification of  claim 11 , wherein the sequence Tags are different.  
     
     
         13 . The method of  claim 9 , wherein the double stranded DNA sequence used in steps b) and e) contains a promoter for different RNA polymerase.  
     
     
         14 . The method of  claim 8 , wherein step d) is performed in a reaction comprising one or more nucleotide analogues.  
     
     
         15 - 21 . (canceled)  
     
     
         22 . The method according to  claim 14 , where the synthesized cRNA is used to measure gene expression.  
     
     
         23 - 24 . (canceled)  
     
     
         25 . The method of  claim 1 , in which the nucleic acids further comprises an affinity tag wherein the affinity tag can be used to purify the ligated nucleic acid molecule.  
     
     
         26 . A method of analyzing nucleic acid comprising: 
 a) hybridizing an oligodeoxyribonucleotide which contains natural and modified nucleotides to an RNA sequence such that the oligodeoxyribonucleotide is complementary to a portion of the RNA;    b) contacting the resulting RNA-DNA heteroduplex with an agent that specifically nicks only the RNA strand; and    c) ligating a DNA sequence to the trimmed RNA 3′ end of step b).    
     
     
         27 . The method of  claim 26 , further comprising producing cRNA according to the method of  claim 8 .  
     
     
         28 . A method of analyzing nucleic acid comprising: 
 a) hybridizing an oligodeoxyribonucleotide which contains natural and modified nucleotides to an RNA sequence such that the oligodeoxyribonucleotide is complementary to a portion of the RNA and that the oligodeoxyribonucleotide hybridises at the poly(A) tail:message junction    b) contacting the resulting RNA-DNA heteroduplex with an agent that specifically nicks only the RNA strand at the poly(A):message junction    c) determining the size of the poly(A) tail.    
     
     
         29 . A kit comprising a nucleic acid sequence that comprises or nucleic acid sequences that comprise a double stranded region and a single stranded region and a DNA ligase.  
     
     
         30 . The kit of  claim 29 , wherein the DNA ligase is T4 DNA ligase.  
     
     
         31 . The kit of  claim 29 , which additionally contains an exonuclease and an RNA polymerase.  
     
     
         32 . The kit of  claim 29 , which additionally contains oligo (dA).  
     
     
         33 . The kit of  claim 29 , wherein the nucleic acid sequence comprises an oligodeoxynucleotide containing natural and modified nucleotides.