Linear amplification of short nucleic acids
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 from the 5′ ends of a multiplicity of target nucleic acids, said method comprising;
forming a reaction mixture comprising a multiplicity of different target nucleic acids, a collection of target-specific primers, and an enzyme that catalyzes target-specific primer extension, wherein the target-specific primers can hybridize to the target nucleic acids about 15-35 nucleotides away from the 5′ end of the target nucleic acids;
annealing the collection of target-specific primers to the multiplicity of target nucleic acids;
extending the multiplicity of target-specific primers with the enzyme that catalyzes target-specific primer extension; and
cycling the reaction mixture for at least 20 cycles between an annealing temperature segment of 10° C.-30° C., an extension segment, and a denaturation temperature segment of 35° C.-60° C.;
thereby linearly amplifying the multiplicity of different short nucleic acid sequences.
2. The method according to claim 1 wherein the multiplicity of different target nucleic acids are messenger RNAs.
3. The method according to claim 1 wherein the collection of target-specific primers comprise stem-loop primers.
4. The method according to claim 1 wherein the annealing temperature is 20° C.-30° C.
5. The method according to claim 1 wherein the denaturation temperature is 40° C.-50° C.
6. The method according to claim 1 wherein 20 to 200 cycles are performed.
7. The method according to claim 1 wherein the annealing segment is 1-2 minutes during each cycle.
8. The method according to claim 1 wherein the denaturation segment is 2-10 seconds during each cycle.
9. The method according to claim 1 wherein at least 100 different target nucleic acids are amplified.
10. The method according to claim 1 wherein the reaction mixture comprises an osmolyte.
11. The method according to claim 10 wherein the osmolyte is betaine, sorbitol, or glycerol.
12. The method according to claim 11 wherein the betaine, sorbitol, or glycerol are present in a total concentration of about 2 molar in the reaction mixture.
13. The method according to claim 1 wherein the enzyme that catalyzes target specific primer extension is a reverse transcriptase.
14. The method according to claim 13 wherein the reverse transcriptase is heat-sensitive, but fails to lose substantial activity in the at least 20 cycles.
15. 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 50 to 250 cycles are performed.
16. A method of linearly amplifying a short nucleic acid sequence from the 5′ end of a target nucleic acid molecule, said method comprising;
forming a reaction mixture comprising a target nucleic acid molecule, a target-specific primer, and an enzyme that catalyzes target-specific primer extension, wherein the target-specific primer can hybridize to the target nucleic acid about 15-35 nucleotides away from the 5′ end of the target nucleic acid molecule;
annealing the target-specific primer to the target nucleic acid;
extending the target-specific primer with the enzyme that catalyzes target specific primer extension;
and
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.;
thereby linearly amplifying the short nucleic acid sequence.
17. The method according to claim 16 wherein the annealing temperature segment is 20° C.-30° C., the denaturation temperature segment of 40° C.-50° C., and 50 to 250 cycles are performed.
18. The method according to claim 17 wherein the reaction mixture comprises an osmolyte, and the osmolyte is betaine, sorbitol, or glycerol.
19. The method according to claim 18 wherein the betaine, sorbitol, or glycerol are present in a total concentration of about 2 molar in the reaction mixture.
20. The method according to claim 16 wherein the target nucleic acid molecule is a messenger RNA.