Hot start reverse transcription by primer design

Abstract

The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.

1 . A method of reducing primer extension products at a low temperature and allowing primer extension at a high temperature comprising; 
 forming a reaction mixture at the low temperature below about 27C, wherein the reaction mixture comprises a target polynucleotide, a primer extending enzyme, and a hot start primer, wherein the hot start primer comprises a loop and a self-complementary stem, wherein a target-specific region of the self-complementary stem comprises at least six nucleotides that are complementary with the target polynucleotide, wherein the target-specific region of the self-complementary stem is hybridized with a quencher region in the self-complementary stem when at the low temperature, and wherein the self-complementary stem is unable to hybridize with the target polynucleotide when at the low temperature;    elevating the temperature of the reaction mixture to a high temperature between 35C-60C, wherein the target-specific region of the self-complementary stem is unhybridized with the quencher region in the self-complementary stem at the high temperature, and wherein the target-specific region hybridizes to the target polynucleotide;    extending the target-specific region of the self-complementary stem with the primer extending enzyme to form a primer extension product; and,    generating a primer extension product at the high temperature but not at the low temperature.    
     
     
         2 . The method according to  claim 1  wherein the primer extending enzyme is a reverse transcriptase.  
     
     
         3 . The method according to  claim 1  wherein the target polynucleotide is a messenger RNA.  
     
     
         4 . The method according to  claim 1  wherein the target polynucleotide is a small non-coding RNA.  
     
     
         5 . The method according to  claim 4  wherein the small non-coding RNA is a micro RNA.  
     
     
         6 . The method according to  claim 1  wherein the loop of the hot start primer comprises 8-24 nucleotides.  
     
     
         7 . The method according to  claim 1  wherein the self-complementary stem of the hot start primer comprises 6-12 nucleotide base-pairs.  
     
     
         8 . The method according to  claim 1  wherein the target-specific region of the hot start primer further comprises nucleotide s present in the loop of the hot start primer.  
     
     
         9 . The method according to  claim 1  wherein the target-specific region of the hot start primer, when hybridized to the quencher region of the hot start primer, forms a blunt-ended structure.  
     
     
         10 . The method according to  claim 1  wherein the high temperature between 35C-60C occurs for between 20 minutes and 40 minutes.  
     
     
         11 . The method according to  claim 1  wherein the primer extension reaction is a reverse transcription reaction, said method further comprising performing a polymerase chain reaction (PCR) on the primer extension product with a forward primer and a reverse primer, wherein the PCR comprises; 
 a denaturizing step performed after the extension reaction;    an annealing step performed after the melting step; and,    an extension step performed after the annealing step.    
     
     
         12 . The method according to  claim 11  wherein the reverse transcription reaction comprises the forward primer.  
     
     
         13 . A composition comprising a target polynucleotide and a hot start primer primer, wherein the hot start primer comprises a self-complementary stem, wherein the self-complementary stem comprises a target-specific region and a quencher region, wherein the target-specific region comprises at least six nucleotides, wherein the target-specific region is substantially unhybridized with the quencher region in the self-complementary stem, and wherein the target-specific region is hybridized with the target polynucleotide.  
     
     
         14 . The composition according to  claim 13  wherein the target polynucleotide is a messenger RNA.  
     
     
         15 . The composition according to  claim 13  wherein the target polynucleotide is a small non-coding RNA.  
     
     
         16 . The composition according to  claim 15  wherein the small non-coding RNA is a micro RNA.  
     
     
         17 . The composition according to  claim 13  wherein the loop of the hot start primer comprises 8-24 nucleotides.  
     
     
         18 . The composition according to  claim 18  wherein the self-complementary stem of the hot start primer comprises 6-12 nucleotide base-pairs.  
     
     
         19 . The composition according to  claim 13  wherein the target-specific region of the hot start primer further comprises at least one nucleotide present in the loop of the hot start primer.  
     
     
         20 . The composition according to  claim 13  wherein the target-specific region of the hot start primer, when hybridized to the quencher region of the hot start primer, forms a blunt-ended structure.  
     
     
         21 . A kit for reducing primer extension products at a low temperature and allowing primer extension at a high temperature comprising; 
 a hot start primer, a primer extending enzyme, a primer extending enzyme buffer, and dNTPs, wherein the hot start primer comprises a loop and a self-complementary stem, wherein the self-complementary stem comprises a target-specific region that is at least six nucleotides in length, wherein the target-specific region is complementary to target polynucleotide, wherein the target-specific region is substantially hybridized to a quencher region when at a temperature of 27C or lower, and wherein the target-specific region when hybridized to the quencher region forms a blunt end structure, a structure with a one nucleotide overlap, or a structure with a two nucleotide overlap.    
     
     
         22 . The kit according to  claim 21  wherein the primer extending enzyme is a reverse transcriptase.  
     
     
         23 . The kit according to  claim 21  wherein the loop of the hot start primer comprises 8-24 nucleotides.  
     
     
         24 . The kit according to  claim 21  wherein the self-complementary stem of the hot start primer comprises 6-12 nucleotide base-pairs.  
     
     
         25 . The kit according to  claim 21  further comprising a forward primer and reagents for performing a PCR.  
     
     
         26 . The kit according to  claim 25  wherein the reagents for performing the PCR are included in a vessel that is the same vessel that contains at least one of the hot start primer, the primer extending enzyme, the primer extending enzyme buffer, and the dNTPs.  
     
     
         27 . The kit according to  claim 21  wherein the target polynucleotide is selected from the group comprising messenger RNA, small non-coding RNA, and micro RNA.