Methods and compositions for amplification of RNA sequences

Abstract

The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

1 - 139  (canceled)  
     
     
         140 . A composition comprising a composite primer, wherein the composite primer comprises an RNA portion and a 3′ DNA portion, and a second primer.  
     
     
         141 . The composition of  claim 140 , wherein the second primer comprises DNA.  
     
     
         142 . The composition of  claim 141 , wherein the second primer is a random primer.  
     
     
         143 . The composition of  claim 140 , wherein the second primer comprises a fragment of the target RNA hybridized to the primer extension product.  
     
     
         144 . The composition of  claim 140 , further comprising an RNA-dependent DNA polymerase.  
     
     
         145 . The composition of  claim 140 , wherein the composite primer further comprises a 5′ region that is not hybridizable to the RNA sequence of interest under conditions which the composite primer hybridizes to the target RNA.  
     
     
         146 . A composition comprising a composite primer and a second primer that comprises a sequence that is not hybridizable to a first primer extension product under conditions which the composite primer hybridizes to the target RNA.  
     
     
         147 . A composition comprising: (a) a composite primer; (b) a second primer; and (c) a propromoter polynucleotide.  
     
     
         148 . The composition of  claim 147 , wherein the second primer is a random primer.  
     
     
         149 . A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3′ DNA portion; and (b) a propromoter polynucleotide.  
     
     
         150 . A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3′ DNA portion; and (b) a second primer extension product, wherein the second primer comprises DNA.  
     
     
         151 . A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3′. DNA portion; and (b) a second primer extension product, wherein the second primer comprises a fragment of the RNA target.  
     
     
         152 . A composition comprising a complex of (a) a cleaved primer extension product, wherein the primer is a composite primer comprising an RNA portion and a 3′ DNA portion; (b) a second primer extension product; and (c) a composite primer that hybridizes to second primer extension product.  
     
     
         153 . The composition of  claim 152 , wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are the same.  
     
     
         154 . The composition of  claim 152 , wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are different.  
     
     
         155 . A reaction mixture comprising (a) a target RNA; (b) a composite primer comprising a 3′ DNA portion and an RNA portion; (c) a second primer; and (d) a DNA polymerase.  
     
     
         156 . The reaction mixture of  claim 155 , further comprising: (e) an enzyme which cleaves RNA from an RNA/DNA hybrid.  
     
     
         157 . The reaction mixture of  claim 155 , further comprising: (e) a propromoter polynucleotide.  
     
     
         158 - 161  (canceled)