Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs

Abstract

The present teachings provide methods, compositions, and kits for quantifying target polynucleotides. In some embodiments, a reverse stem-loop ligation probe is ligated to the 3′ end of a target polynucleotide, using a ligase that can ligate the 3′ end of RNA to the 5′ end of DNA using a DNA template, such as T4 DNA ligase. Following digestion to form an elongated target polynucleotide with a liberated end, a reverse transcription reaction can be performed, followed by a PCR. In some embodiments, the methods of the present teachings can discriminate between polymorphic polynucleotides that vary by as little as one nucleotide.

1 . A method of amplifying a target polynucleotide, the method comprising:
 forming a first reaction complex comprising a reverse stem-loop ligation probe hybridized to the target polynucleotide, wherein the reverse stem-loop ligation probe comprises a loop, a stem, and a 3′ target-specific portion;   ligating the reverse stem-loop ligation probe to the target polynucleotide to form an elongated target polynucleotide;   removing the hybridized 3′ target-specific portion from the elongated target polynucleotide to form an elongated target polynucleotide with a liberated end;   forming a second reaction complex comprising a reverse primer hybridized to the liberated end of the elongated target polynucleotide;   extending the reverse primer to form a first strand, wherein the first strand is hybridized to the elongated target polynucleotide to form a double stranded complex; and,   amplifying the double stranded complex.   
     
     
         2 . The method according to  claim 1  wherein the reverse stem-loop ligation probe comprises at least one uracil residue, wherein the removing comprises enzymatic degradation of the uracil. 
     
     
         3 . The method according to  claim 1  wherein the target polynucleotide is an RNA molecule, wherein the reverse stem-loop ligation probe is a DNA molecule, and wherein the ligase is T4 DNA ligase. 
     
     
         4 . The method according to  claim 1  wherein the liberated end of the elongated target polynucleotide corresponds to the loop of the reverse stem-loop ligation probe. 
     
     
         5 . The method according to  claim 1  wherein the amplifying comprises a PCR, wherein the PCR comprises a forward primer, and wherein the forward primer comprises a target specific portion and a 5′ tail. 
     
     
         6 . The method according to  claim 1  wherein the amplifying is a real-time PCR. 
     
     
         7 . The method according to  claim 6  wherein the real-time PCR comprises a nucleic acid detector probe, wherein the nucleic acid detector probe comprises a sequence complementary to the stem of the reverse stem-loop ligation probe, or comprises a sequence complementary to the complement of the stem of the reverse stem-loop ligation probe. 
     
     
         8 . The method according to  claim 7  wherein the nucleic acid detector probe further comprises a sequence complementary to the target polynucleotide, or comprises a sequence complementary to the complement of the target polynucleotide. 
     
     
         9 . The method according to  claim 7  wherein the detector probe is a 5′ nuclease cleavable probe. 
     
     
         10 . The method according to  claim 1  wherein the 3′ target-specific portion of the reverse stem-loop ligation probe comprises an extension blocker. 
     
     
         11 . The method according to  claim 10  wherein the extension blocker is an amine group. 
     
     
         12 . A kit for amplifying at least three target polynucleotides, the kit comprising, at least three species of reverse stem-loop ligation probes, wherein the at least three species of reverse stem-loop ligation probes vary from each other in the sequence of the 3′ target-specific portion, wherein the at least three species of reverse stem-loop ligation probes vary from each other in the sequence of their stem, and wherein the at least three species of reverse stem-loop ligation probes vary from each other in the sequence of their loop. 
     
     
         13 . The kit according to  claim 12 , wherein the at least three species of reverse stem-loop ligation probes comprise an extension blocker, a degradable nucleotide, or both an extension blocker and a degradable nucleotide. 
     
     
         14 . The kit-according to  claim 12  further comprising a reverse transcriptase. 
     
     
         15 . The kit according to  claim 12  further comprising a polymerase. 
     
     
         16 . The kit according to  claim 12  further comprising a reverse primer, a forward primer, and a detector probe. 
     
     
         17 - 23 . (canceled) 
     
     
         24 . A reaction composition comprising at least three species of reverse stem-loop ligation probes, wherein the at least three species of reverse stem-loop ligation probes vary from each other in the sequence of the 3′ target-specific portion, wherein the at least three species of reverse stem-loop, ligation probes vary from each other in the sequence of their stem, and wherein the at least three species reverse stem-loop ligation probes vary from each other in the sequence of their loop. 
     
     
         25 . The reaction composition according to  claim 24  wherein the at least three species of reverse stem-loop ligation probes each comprise an extension blocker, a degradable nucleotide, or both an extension blocker and a degradable nucleotide. 
     
     
         26 . The reaction composition according to  claim 24  wherein the 3′ target-specific portion comprises the extension blocker, and wherein the stem comprises the degradable nucleotide. 
     
     
         27 . The reaction composition according to  claim 24  wherein the 3′ target-specific portion of each of the at least three species of reverse stem-loop ligation probes is complementary to an ShRNA-derived siRNA. 
     
     
         28 - 31 . (canceled)