Methods, Compositions, and Kits Comprising Linker Probes for Quantifying Polynucleotides

Abstract

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

1 . A method for detecting a micro RNA (miRNA) comprising;
 hybridizing the miRNA and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA;   extending the linker probe to form an extension reaction product;   amplifying the extension reaction product to form an amplification product; and,   detecting the miRNA.   
     
     
         2 . The method according to  claim 1  wherein the amplification reaction is a polymerase chain reaction, wherein the amplification reaction comprises a forward primer that corresponds to the miRNA, and a reverse primer that corresponds to the linker probe. 
     
     
         3 . The method according to  claim 1  wherein the miRNA is 18-25 ribonucleotides in length. 
     
     
         4 . The method according to  claim 1  wherein the amplification reaction comprises a detector probe. 
     
     
         5 . The method according to  claim 4  wherein the detector probe comprises a nucleotide of the linker probe in the amplification product or a nucleotide of the linker probe complement in the amplification product. 
     
     
         6 . The method according to  claim 4  wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product. 
     
     
         7 . The method according to  claim 4  wherein the detector probe comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product. 
     
     
         8 . The method according to  claim 4  wherein the detector probe comprises a nucleotide of a region upstream from the 3′ end region of the miRNA in the amplification product or a nucleotide of a region upstream from the 3′ end region of the miRNA complement in the amplification product. 
     
     
         9 . The method according to  claim 4  wherein the detector probe is a 5′-nuclease cleavable probe. 
     
     
         10 . The method according to  claim 9  wherein the 5′-nuclease cleavable probe comprises FAM. 
     
     
         11 . The method according to  claim 9  wherein the 5′-nuclease cleavable probe comprises VIC. 
     
     
         12 . The method according to  claim 4  wherein the detector probe comprises peptide nucleic acid (PNA). 
     
     
         13 . The method according to  claim 12  wherein the PNA probe comprises FAM. 
     
     
         14 . The method according to  claim 12  wherein the PNA probe comprises VIC. 
     
     
         15 . The method according to  claim 4  wherein the detector probe comprises locked nucleic acid (LNA). 
     
     
         16 . The method according to  claim 4  wherein the detector probe comprises a universal base. 
     
     
         17 . The method according to  claim 4  wherein the detector probe is an intercalating dye. 
     
     
         18 . The method according to  claim 1  wherein the extending is a reverse transcription reaction comprising a reverse transcriptase. 
     
     
         19 . The method according to  claim 1  wherein the stem of the linker probe comprises 12-16 base-pairs. 
     
     
         20 . The method according to  claim 19  wherein the stem of the linker probe comprises 14 base-pairs. 
     
     
         21 . The method according to  claim 1  wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides. 
     
     
         22 . The method according to  claim 1  wherein the loop corresponds to a universal reverse primer portion. 
     
     
         23 . The method according to  claim 1  wherein the loop comprises 14-18 nucleotides. 
     
     
         24 . The method according to  claim 23  wherein the loop comprises 16 nucleotides. 
     
     
         25 . The method according to  claim 4  wherein the Tm of the detector probe is 63-69 C. 
     
     
         26 . A method for detecting a target polynucleotide comprising;
 hybridizing the target polynucleotide and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide;   extending the linker probe to form an extension reaction product;   amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product; and,   detecting the target polynucleotide.   
     
     
         27 . The method according to  claim 26  wherein the amplification reaction is a polymerase chain reaction, wherein the amplification reaction comprises a forward primer that corresponds to the target polynucleotide, and a reverse primer that corresponds to the linker probe. 
     
     
         28 . The method according to  claim 26  wherein the target polynucleotide is a micro RNA (miRNA). 
     
     
         29 . The method according to  claim 26  wherein the detector probe comprises a nucleotide of the 3′ end region of the target polynucleotide in the amplification product or a nucleotide of the 3′ end region of the target polynucleotide complement in the amplification product. 
     
     
         30 . The method according to  claim 26  wherein the detector probe comprises a nucleotide of a region upstream from the 3′ end region of the target polynucleotide in the amplification product or a nucleotide of a region upstream from the 3′ end region of the target polynucleotide complement in the amplification product. 
     
     
         31 . The method according to  claim 26  wherein the detector probe is a 5′-nuclease cleavable probe. 
     
     
         32 . The method according to  claim 31  wherein the 5′-nuclease cleavable probe comprises FAM. 
     
     
         33 . The method according to  claim 31  wherein the 5′-nuclease cleavable probe comprises VIC. 
     
     
         34 . The method according to  claim 26  wherein the detector probe comprises peptide nucleic acid (PNA). 
     
     
         35 . The method according to  claim 34  wherein the PNA probe comprises FAM. 
     
     
         36 . The method according to  claim 34  wherein the PNA probe comprises VIC. 
     
     
         37 . The method according to  claim 26  wherein the detector probe comprises locked nucleic acid (LNA). 
     
     
         38 . The method according to  claim 26  wherein the detector probe comprises a universal base. 
     
     
         39 . The method according to  claim 26  the extending is a reverse transcription reaction comprising a reverse transcriptase. 
     
     
         40 . The method according to  claim 26  wherein the stem of the linker probe comprises 12-16 base-pairs. 
     
     
         41 . The method according to  claim 40  wherein the stem of the linker probe comprises 14 base-pairs. 
     
     
         42 . The method according to  claim 26  wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides. 
     
     
         43 . The method according to  claim 26  wherein the loop further comprises a universal reverse primer portion. 
     
     
         44 . The method according to  claim 26  wherein the loop comprises 14-18 nucleotides. 
     
     
         45 . The method according to  claim 44  wherein the loop comprises 16 nucleotides. 
     
     
         46 . The method according to  claim 26  wherein the Tm of the detector probe is 63-69 C. 
     
     
         47 . A method for detecting a miRNA molecule comprising;
 hybridizing the miRNA molecule and a linker probe,
 wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide; 
   extending the linker probe to form an extension reaction product;   amplifying the extension reaction product in the presence of a detector probe to form an amplification product, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and,   detecting the miRNA molecule.   
     
     
         48 . The method according to  claim 47  wherein the amplification reaction is a polymerase chain reaction, wherein the amplification reaction comprises a forward primer that corresponds to the miRNA, and a reverse primer that corresponds to the linker probe. 
     
     
         49 . The method according to  claim 47  wherein the miRNA is 18-25 ribonucleotides in length. 
     
     
         50 . The method according to  claim 47  wherein the detector probe is a 5′-nuclease cleavable probe. 
     
     
         51 . The method according to  claim 50  wherein the 5′-nuclease cleavable probe comprises FAM. 
     
     
         52 . The method according to  claim 50  wherein the 5′-nuclease cleavable probe comprises VIC. 
     
     
         53 . The method according to  claim 47  wherein the detector probe comprises peptide nucleic acid (PNA). 
     
     
         54 . The method according to  claim 53  wherein the PNA probe comprises FAM. 
     
     
         55 . The method according to  claim 53  wherein the PNA probe comprises VIC. 
     
     
         56 . The method according to  claim 47  wherein the detector probe comprises locked nucleic acid (LNA). 
     
     
         57 . The method according to  claim 47  wherein the detector probe comprises a universal base. 
     
     
         58 . The method according to  claim 47  wherein the extending is a reverse transcription reaction comprising a reverse transcriptase. 
     
     
         59 . The method according to  claim 47  wherein the stem of the linker probe comprises 12-16 base-pairs. 
     
     
         60 . The method according to  claim 59  wherein the stem of the linker probe comprises 14 base-pairs. 
     
     
         61 . The method according to  claim 47  wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides. 
     
     
         62 . The method according to  claim 47  wherein the loop further comprises a universal reverse primer portion. 
     
     
         63 . The method according to  claim 47  wherein the loop comprises 14-18 nucleotides. 
     
     
         64 . The method according to  claim 63  wherein the loop comprises 16 nucleotides. 
     
     
         65 . The method according to  claim 47  wherein the Tm of the detector probe is 63-69 C. 
     
     
         66 . A method for detecting two different miRNAs from a single hybridization reaction comprising;
 hybridizing a first miRNA and a first linker probe, and a second miRNA and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first miRNA, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second miRNA;   extending the first linker probe and the second linker probe to form extension reaction products;   dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product, and a second amplification reaction to form a second amplification reaction product,   wherein a primer in the first amplification reaction corresponds with the first miRNA and not the second miRNA, and a primer in the second amplification reaction corresponds with the second miRNA and not the first miRNA,   wherein a first detector probe in the first amplification reaction differs from a second detector probe in the second amplification reaction,   wherein the first detector probe comprises a nucleotide of the first linker probe stem of the amplification product or a nucleotide of the first linker probe stem complement in the first amplification product,   wherein the second detector probe comprises a nucleotide of the second linker probe stem of the amplification product or a nucleotide of the second linker probe stem complement in the amplification product; and,   detecting the two different miRNAs.   
     
     
         67 . The method according to  claim 66  wherein the first amplification reaction is a first polymerase chain reaction and the second amplification reaction is a second polymerase chain reaction;
 wherein the first polymerase chain reaction comprises a forward primer that corresponds to the first miRNA, and a reverse primer that corresponds to the linker probe, 
 wherein the second polymerase chain reaction comprises a forward primer that corresponds to the second miRNA, and a reverse primer that corresponds to the linker probe, 
 wherein the reverse primer in the first polymerase chain reaction and the reverse primer in the second polymerase chain reaction are a universal reverse primer. 
 
     
     
         68 . The method according to  claim 66  wherein the first miRNA and/or the second miRNA is 18-25 ribonucleotides in length. 
     
     
         69 . The method according to  claim 66  wherein the first detector probe and/or the second detector probe is a 5′-nuclease cleavable probe. 
     
     
         70 . The method according to  claim 69  wherein the first detector probe and/or the second detector probe comprises FAM. 
     
     
         71 . The method according to  claim 69  wherein the first detector probe and/or the second detector probe comprises VIC. 
     
     
         72 . The method according to  claim 66  wherein the first detector probe and/or the second detector probe comprises peptide nucleic acid (PNA). 
     
     
         73 . The method according to  claim 72  wherein first detector probe and/or the second detector probe comprises FAM. 
     
     
         74 . The method according to  claim 72  wherein the first detector probe and/or the second detector probe comprises VIC. 
     
     
         75 . The method according to  claim 66  wherein the first detector probe and/or the second detector probe comprises locked nucleic acid (LNA). 
     
     
         76 . The method according to  claim 66  wherein the first detector probe and/or the second detector probe comprises a universal base. 
     
     
         77 . The method according to  claim 66  wherein the extending is a reverse transcription reaction comprising a reverse transcriptase. 
     
     
         78 . The method according to  claim 66  wherein the stem of the first linker probe and/or the second linker probe comprises 12-16 base-pairs. 
     
     
         79 . The method according to  claim 78  wherein the stem of the first linker probe and/or the second linker probe comprises 14 base-pairs. 
     
     
         80 . The method according to  claim 66  wherein the 3′ target specific portion of the first linker probe and/or the second linker probe comprises 5-8 nucleotides. 
     
     
         81 . The method according to  claim 66  wherein the loop of the first linker probe and/or the second linker probe further comprises a universal reverse primer portion. 
     
     
         82 . The method according to  claim 66  wherein the loop of the first linker probe and/or the second linker probe comprises 14-18 nucleotides. 
     
     
         83 . The method according to  claim 82  wherein the loop of the first linker probe and/or the second linker probe comprises 16 nucleotides. 
     
     
         84 . The method according to  claim 66  wherein the Tm of the first detector probe and/or the second detector probe is 63-69 C. 
     
     
         85 . A method for detecting two different target polynucleotides from a single hybridization reaction comprising;
 hybridizing a first target polynucleotide and a first linker probe, and a second target polynucleotide and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first target polynucleotide, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second target polynucleotide;   extending the first linker probe and the second linker probe to form extension reaction products;   dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product and a second amplification reaction to form a second amplification reaction product; and,   detecting the two different miRNA molecules.   
     
     
         86 . The method according to  claim 85  wherein the first amplification reaction is a first polymerase chain reaction and the second amplification reaction is a second polymerase chain reaction;
 wherein the first polymerase chain reaction comprises a forward primer that corresponds to the first target polynucleotide, and a reverse primer that corresponds to the linker probe, 
 wherein the second polymerase chain reaction comprises a forward primer that corresponds to the second target polynucleotide, and a reverse primer that corresponds to the linker probe, 
 wherein the reverse primer in the first polymerase chain reaction and the reverse primer in the second polymerase chain reaction are a universal reverse primer. 
 
     
     
         87 . The method according to  claim 85  wherein the target polynucleotide is a micro RNA (miRNA). 
     
     
         88 . The method according to  claim 85  wherein the first amplification reaction comprises a first detector probe and/or the second amplification reaction comprises a second detector probe. 
     
     
         89 . The method according to  claim 88  wherein the first detector probe corresponds with a nucleotide of the first linker probe in the first amplification product or a nucleotide of the first linker probe complement in the first amplification product, and/or the second detector probe corresponds with a nucleotide of the second linker probe in the second amplification product or a nucleotide of the second linker probe complement in the second amplification product 
     
     
         90 . The method according to  claim 88  wherein the first detector probe comprises a nucleotide of the first linker probe stem of the first amplification product or a nucleotide of the first linker probe stem complement in the first amplification product, and/or the second detector probe comprises a nucleotide of the second linker probe stem in the second amplification product or a nucleotide of the second linker probe stem complement in the second amplification product. 
     
     
         91 . The method according to  claim 88  wherein the first detector probe comprises a nucleotide of the 3′ end region of the first target polynucleotide in the first amplification product or a nucleotide of the 3′ end region of the first target polynucleotide complement in the first amplification product, and/or the second detector probe comprises a nucleotide of the 3′ end region of the second target polynucleotide in the second amplification product or a nucleotide of the 3′ end region of the second target polynucleotide complement in the second amplification product. 
     
     
         92 . The method according to  claim 88  wherein the first detector probe corresponds with a nucleotide of a region upstream from the 3′ end region of the first target polynucleotide in the first amplification product or a nucleotide of a region upstream from the 3′ end region of the first target polynucleotide complement in the first amplification product, and/or the second detector probe corresponds with a nucleotide of a region upstream from the 3′ end region of the second target polynucleotide in the second amplification product or a nucleotide of a region upstream from the 3′ end region of the second target polynucleotide complement in the second amplification product. 
     
     
         93 . The method according to  claim 85  wherein the first target polynucleotide and/or the second target polynucleotide is 18-25 ribonucleotides in length. 
     
     
         94 . The method according to  claim 88  wherein the first detector probe and/or second detector probe is a 5′-nuclease cleavable probe. 
     
     
         95 . The method according to  claim 94  wherein the first detector probe and/or second detector probe comprises FAM. 
     
     
         96 . The method according to  claim 94  wherein the first detector probe and/or second detector probe comprises VIC. 
     
     
         97 . The method according to  claim 88  wherein the first detector probe and/or second detector probe comprises peptide nucleic acid (PNA). 
     
     
         98 . The method according to  claim 97  wherein first detector probe and/or second detector probe comprises FAM. 
     
     
         99 . The method according to  claim 97  wherein the first detector probe and/or second detector probe comprises VIC. 
     
     
         100 . The method according to  claim 88  wherein the first detector probe and/or the second detector probe comprises locked nucleic acid (LNA). 
     
     
         101 . The method according to  claim 88  wherein the first detector probe and/or the second detector probe comprises a universal base. 
     
     
         102 . The method according to  claim 85  wherein the extending is a reverse transcription reaction comprising a reverse transcriptase. 
     
     
         103 . The method according to  claim 85  wherein the stem of the first linker probe and/or the second linker probe comprises 12-16 base-pairs. 
     
     
         104 . The method according to  claim 103  wherein the stem of the first linker probe and/or the second linker probe comprises 14 base-pairs. 
     
     
         105 . The method according to  claim 85  wherein the 3′ target specific portion of the first linker probe and/or the second linker probe comprises 5-8 nucleotides. 
     
     
         106 . The method according to  claim 85  wherein the loop of the first linker probe and/or the second linker probe comprises a universal reverse primer portion. 
     
     
         107 . The method according to  claim 85  wherein the loop of the first linker probe and/or the second linker probe comprises 14-18 nucleotides. 
     
     
         108 . The method according to  claim 107  wherein the loop of the first linker probe and/or the second linker probe comprises 16 nucleotides. 
     
     
         109 . The method according to  claim 88  wherein the Tm of the first detector probe and/or second detector probe is 63-69 C. 
     
     
         110 . A method for detecting a miRNA molecule from a cell lysate comprising;
 hybridizing the miRNA molecule from the cell lysate with a linker probe,
 wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA, 
   extending the linker probe to form an extension reaction product;   amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem of the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and,   detecting the miRNA molecule.   
     
     
         111 . The method according to  claim 110 , wherein the cell lysate comprises;
 treating cells with a lysis buffer, wherein the lysis buffer comprises,
 10 mM Tris-HCl, pH 8.0; 
 0.02% Sodium Azide; and, 
 0.03% Tween-20. 
   
     
     
         112 . A kit comprising;
 a reverse transcriptase and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion corresponds to a miRNA.   
     
     
         113 . The kit according to  claim 112  further comprising a DNA polymerase. 
     
     
         114 . The kit according to  claim 112  further comprising a primer pair. 
     
     
         115 . The kit according to  claim 114  wherein the primer pair comprises,
 a forward primer specific for a miRNA, and, 
 a universal reverse primer, wherein the universal reverse primer comprises a nucleotide of the loop of the linker probe. 
 
     
     
         116 . The kit according to  claim 112  comprising a plurality of primer pairs, wherein each primer pair is in one reaction vessel of a plurality of reaction vessels. 
     
     
         117 . The kit according to  claim 112  further comprising a detector probe. 
     
     
         118 . The kit according to  claim 117  wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product.