Linear amplification of short nucleic acids

Abstract

The present teachings provide novel methods for amplifying short nucleic acids. In some embodiments, the present teachings provide novel methods for linearly amplifying a collection of micro RNAs by using temperature cycling during a reverse transcription reaction. The cycling can comprise at least 20 cycles of an annealing temperature segment of 10° C.-30° C., and a denaturation temperature segment of 35° C.-60° C. In some embodiments, the temperature cycled reaction can comprise an osmolyte.

1 . A method of linearly amplifying a multiplicity of different short nucleic acid sequences, wherein the multiplicity of short nucleic acid sequences are 15-35 nucleotides in length, said method comprising;
 forming a reaction mixture comprising a multiplicity of different short nucleic acids, a multiplicity of target-specific primers, and an enzyme that catalyzes target-specific primer extension;   annealing the collection of target-specific primers to the multiplicity of different short nucleic acids;   extending the multiplicity of target-specific primers with the enzyme that catalyzes target-specific primer extension;   cycling the reaction mixture for at least 20 cycles between an annealing temperature segment of 10° C.-30° C., and a denaturation temperature segment of 35° C.-60° C.; and,   linearly amplifying the multiplicity of different short nucleic acid sequences.   
   
   
       2 . The method according to  claim 1  wherein the multiplicity of different short nucleic acid sequences comprise micro RNAs. 
   
   
       3 . The method according to  claim 1  wherein the reverse transcriptase is heat-sensitive, but fails to lose substantial activity in the at least 20 cycles. 
   
   
       4 . The method according to  claim 1  wherein the collection of target-specific primers comprise stem-loop primers. 
   
   
       5 . The method according to  claim 1  wherein the annealing temperature is 20° C.-30° C. 
   
   
       6 . The method according to  claim 1  wherein the annealing temperature is 24° C.-28° C. 
   
   
       7 . The method according to  claim 1  wherein the denaturation temperature is 40° C.-50° C. 
   
   
       8 . The method according to  claim 1  wherein the denaturation temperature is 42° C.-48° C. 
   
   
       9 . The method according to  claim 1  wherein 100-200 cycles are performed. 
   
   
       10 . The method according to  claim 1  wherein the annealing segment is 1-2 minutes during each cycle. 
   
   
       11 . The method according to  claim 1  wherein the denaturation segment is 2-10 seconds during each cycle. 
   
   
       12 . The method according to  claim 1  wherein at least 100 different short nucleic acid sequences are amplified. 
   
   
       13 . The method according to  claim 1  wherein the reaction mixture comprises an osmolyte. 
   
   
       14 . The method according to  claim 13  wherein the osmolyte is betaine, sorbitol, or glycerol. 
   
   
       15 . The method according to  claim 14  wherein the betaine, sorbitol, or glycerol are present in a total concentration of about 2 molar in the reaction mixture. 
   
   
       16 . The method according to  claim 1  wherein the enzyme that catalyzes target-specific primer extension is a reverse transcriptase. 
   
   
       17 . The method according to  claim 1  wherein the annealing temperature segment is 20° C.-30° C., the denaturation temperature segment of 40° C.-50° C., and at least 50 cycles are performed. 
   
   
       18 . A method of linearly amplifying a short nucleic acid sequence, wherein the short nucleic acid sequence is 15-35 nucleotides in length, said method comprising;
 forming a reaction mixture comprising the short nucleic acid, a target-specific primer, and an enzyme that catalyzes target-specific primer extension;   annealing the target-specific primer to the short nucleic acid;   extending the target-specific primer with the enzyme that catalyzes target-specific primer extension;   cycling the reaction mixture for at least 20 cycles between an annealing temperature segment of 10° C.-30° C., and a denaturation temperature segment of 35° C.-60° C.; and,   linearly amplifying the short nucleic acid sequence.   
   
   
       19 . The method according to  claim 18  wherein the annealing temperature segment is 20° C.-30° C., the denaturation temperature segment of 40° C.-50° C., and at least 50 cycles are performed. 
   
   
       20 . The method according to  claim 19  wherein the reaction mixture comprises an osmolyte, and the osmolyte is betaine, sorbitol, or glycerol.