Methods and compositions for the amplification of mrna

Abstract

Provided herein are compositions and methods for the synthesis, amplification, and in vitro transcription of full-length cDNA, or cDNA fragments. Methods are provided for reverse transcription of RNA and amplification for in vitro transcription. Further provided are method for loading of dendritic cells with the RNA and homologous lysate for immune stimulation.

1 . An oligonucleotide comprising the sequence of SEQ ID NO: 1. 
     
     
         2 - 9 . (canceled) 
     
     
         10 . A method of synthesizing a first cDNA strand from a template mRNA comprising the steps of:
 a) hybridizing a first primer to the template mRNA, wherein the first primer comprises a nucleic acid sequence of SEQ ID NO: 2;   b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang;   c) hybridizing a second primer to the oligo(C) overhang of the partial first cDNA strand, wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1; and   d) extending the partial first cDNA strand from the oligo(C) overhang using the second primer as the template, thereby generating a first cDNA strand.   
     
     
         11 . The method of  claim 10 , further comprising:
 e) separating the template mRNA and first cDNA strand;   f) hybridizing a third primer to the first cDNA strand; and   g) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA.   
     
     
         12 . The method of  claim 11 , wherein the second primer comprises a nucleic acid sequence of SEQ ID NO: 1. 
     
     
         13 . The method of  claim 11 , wherein the third primer comprises a nucleic acid sequence of SEQ ID NO: 3. 
     
     
         14 . The method of  claim 11 , wherein the template mRNA is obtained from a sample. 
     
     
         15 . The method of  claim 14 , wherein the sample is a tumor sample. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . A method of preparing an RNA by transcribing a cDNA synthesized from an mRNA, the method comprising the steps of:
 a) in an in vitro reaction mixture, hybridizing a first primer to the mRNA, wherein the first primer comprises both SEQ ID NO: 2 and a poly(T) sequence whereby the first primer anneals to the mRNA;   b) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang;   c) hybridizing a second primer comprising SEQ ID NO: 1 to the oligo(C) overhang of the partial first cDNA strand;   d) extending the partial first cDNA strand in the 3′ direction from the oligo(C) overhang using the second primer as a template, thereby generating a first cDNA strand;   e) hybridizing a third primer that comprises an RNA promoter to the first cDNA strand;   f) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA; and   g) synthesizing RNA from the double stranded cDNA.   
     
     
         19 . The method of  claim 18 , wherein the synthesizing RNA from the double stranded cDNA comprises in vitro transcription. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The method of  claim 18 , wherein the method further comprises capping the RNA. 
     
     
         24 . The method of  claim 18 , further comprising amplifying the double stranded cDNA before step g). 
     
     
         25 - 26 . (canceled) 
     
     
         27 . The method of  claim 24 , wherein amplifying the double stranded cDNA comprises adding a DNA dependent DNA polymerase, a fourth primer, and a fifth primer and amplifying the cDNA by polymerase chain reaction. 
     
     
         28 . The method of  claim 27 , wherein the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 2. 
     
     
         29 . The method of  claim 27 , wherein the fifth primer comprises the nucleic acid sequence of SEQ ID NO: 3. 
     
     
         30 . The method of  claim 24 , further comprising in vitro transcribing the amplified cDNA to generate sense-strand amplified mRNA. 
     
     
         31 - 34 . (canceled) 
     
     
         35 . The method of  claim 30 , further comprising capping the amplified RNA. 
     
     
         36 . A method for transducing a dendritic cell population comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, wherein the nucleic acid comprises RNA generated by the method of  claim 18 . 
     
     
         37 . The method of  claim 36 , further comprising contacting the dendritic cell population with a tumor cell lysate. 
     
     
         38 . (canceled) 
     
     
         39 . A method for providing an immune response in a subject having a diseased cell population comprising:
 a) obtaining a primed dendritic cell population produced by the method according to  claim 37 ; and   b) administering an effective amount of the primed dendritic cell population to the subject.   
     
     
         40 - 44 . (canceled) 
     
     
         45 . A kit comprising an oligonucleotide primer of  claim 1 , an oligonucleotide primer of SEQ ID NO:2 and/or an oligonucleotide primer of SEQ ID NO:3. 
     
     
         46 - 51 . (canceled)