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 polynucleoitides 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 11  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 go  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 . A kit for amplifying at least two different target polynucleotides in a family of target polynucleotides, wherein the at least two different target polynucleotides in the family of target polynucleotides vary in no more than two nucleotides in the last three nucleotides at their 3′ end regions, the kit comprising at least two species of reverse stem-loop ligation probes, wherein the two species of reverse stem-loop ligation probes vary from each other in their 3′ target-specific portions.  
     
     
         18 . The kit according to  claim 17  wherein the at least two 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.  
     
     
         19 . The kit according go  claim 17  wherein the at least two species of reverse stem-loop ligation probes comprise an extension blocker, a degradable nucleotide, or both an extension blocker and a degradable nucleotide.  
     
     
         20 . The kit according to  claim 17  further comprising a reverse transcriptase.  
     
     
         21 . The kit according to  claim 17  further comprising a polymerase.  
     
     
         22 . The kit according to  claim 17  further comprising a reverse primer, a forward primer, and a detector probe.  
     
     
         23 . The kit according to  claim 17  wherein the family of target polynucleotides are ShRNA-derived siRNAs.  
     
     
         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 go  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 . A reaction composition comprising at least three species of reverse stem-loop primers, wherein the at least three species of reverse stem-loop primers vary from each other in the sequence of the 3′ target-specific portion, wherein the at least three species reverse stem-loop primers vary from each other in the sequence of their stem, and wherein the at least three species of reverse stem-loop primers vary from each other in the sequence of their loop.  
     
     
         29 . The reaction composition according go  claim 28  wherein the at least three species of reverse stem-loop primers each comprise a degradable nucleotide.  
     
     
         30 . The reaction composition according to  claim 28  wherein the stem comprises the degradable nucleotide.  
     
     
         31 . The reaction composition according to  claim 28  wherein the 3′ target-specific portion of each of the at least three reverse stem-loop primers is complementary to an ShRNA-derived siRNA.