US7575863B2
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Methods, compositions, and kits comprising linker probes for quantifying polynucleotides
Assignee: Applied Biosystems LLC
•
Filing: 2004-09-21
•
Granted: 2009-08-18
•
Expires: 2024-09-21
Abstract
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.
Inventors
- 👤 Caifu Chen
- 👤 Dana Ridzon
- 👤 Zhaohui Zhou
- 👤 Kai Qin Lao
- 👤 Neil A. Straus
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 15:33
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6844; C12Q1/686; C07; C07H; C07H21/00; C07H21/04; C12N; C12N9/00; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q2525/00; C12Q2525/30; C12Q2525/301; C12Q2600/00; C12Q2600/16; C12Q2600/178
Claims Analysis
Total Claims: 111
Claim 1
Independent Claim
154 words
A method for detecting a micro RNA (miRNA), wherein the miRNA is 18-25 ribonucleotides in length, 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 a 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in a polymerase chain reaction comprising a detector probe and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, to form an amplification product, with the proviso that the linker probe is a different molecule from the detector probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence of the linker probe stem in the amplification product; and, detecting the miRNA.
Claim 2
Independent Claim
154 words
A method for detecting a micro RNA (miRNA), wherein the miRNA is 18-25 ribonucleotides in length, 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 a 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in a polymerase chain reaction comprising a detector probe and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, to form an amplification product, with the proviso that the linker probe is a different molecule from the detector probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence of the linker probe stem in the amplification product; and, detecting the miRNA.
Claim 3
Dependent Claim
27 words
The method according to claim 1 wherein the polymerase chain reaction comprises a forward primer that hybridizes to the miRNA or a complementary sequence to the miRNA.
Claim 4
Dependent Claim
47 words
The method according to claim 1 wherein the detector probe hybridizes to a nucleotide of the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the miRNA in the amplification product.
Claim 5
Dependent Claim
55 words
The method according to claim 1 wherein the detector probe hybridizes to a nucleotide of a region upstream from the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to a region upstream from the 3′ end region of the miRNA in the amplification product.
Claim 6
Dependent Claim
15 words
The method according to claim 1 wherein the detector probe is a 5′-nuclease cleavable probe.
Claim 7
Dependent Claim
13 words
The method according to claim 5 wherein the 5′-nuclease cleavable probe comprises FAM.
Claim 8
Dependent Claim
13 words
The method according to claim 5 wherein the 5′-nuclease cleavable probe comprises VIC.
Claim 9
Dependent Claim
15 words
The method according to claim 1 wherein the detector probe comprises peptide nucleic acid (PNA).
Claim 10
Dependent Claim
12 words
The method according to claim 8 wherein the PNA probe comprises FAM.
Claim 11
Dependent Claim
12 words
The method according to claim 8 wherein the PNA probe comprises VIC.
Claim 12
Dependent Claim
15 words
The method according to claim 1 wherein the detector probe comprises locked nucleic acid (LNA).
Claim 13
Dependent Claim
14 words
The method according to claim 1 herein the detector probe comprises a universal base.
Claim 14
Dependent Claim
18 words
The method according to claim 1 wherein the extending is a reverse transcription reaction comprising a reverse transcriptase.
Claim 15
Dependent Claim
16 words
The method according to claim 1 wherein the stem of the linker probe comprises 12-16 base-pairs.
Claim 16
Dependent Claim
16 words
The method according to claim 14 wherein the stem of the linker probe comprises 14 base-pairs.
Claim 17
Dependent Claim
19 words
The method according to claim 1 wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides.
Claim 18
Dependent Claim
16 words
The method according to claim 1 wherein the loop corresponds to a universal reverse primer portion.
Claim 19
Dependent Claim
12 words
The method according to claim 1 wherein the loop comprises 14-18 nucleotides.
Claim 20
Dependent Claim
12 words
The method according to claim 18 wherein the loop comprises 16 nucleotides.
Claim 21
Dependent Claim
16 words
The method according to claim 1 wherein the Tm of the detector probe is 63-69 C.
Claim 22
Independent Claim
175 words
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 a 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 amplification is a polymerase chain reaction, wherein the amplification comprises a forward primer that hybridizes to the target polynucleotide or a complementary sequence to the target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the target polynucleotide.
Claim 23
Independent Claim
175 words
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 a 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 amplification is a polymerase chain reaction, wherein the amplification comprises a forward primer that hybridizes to the target polynucleotide or a complementary sequence to the target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the target polynucleotide.
Claim 24
Dependent Claim
15 words
The method according to claim 21 wherein the target polynucleotide is a micro RNA (miRNA).
Claim 25
Dependent Claim
49 words
The method according to claim 21 wherein the detector probe hybridizes to a nucleotide of the 3′ end region of the target polynucleotide in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the target polynucleotide in the amplification product.
Claim 26
Dependent Claim
58 words
The method according to claim 21 wherein the detector probe hybridizes to a nucleotide of a region upstream from the 3′ end region of the target polynucleotide in the amplification product or hybridizes to a nucleotide of a complementary sequence to a region up stream from the 3′ end region of the target polynucleotide in the amplification product.
Claim 27
Dependent Claim
15 words
The method according to claim 21 wherein the detector probe is a 5′-nuclease cleavable probe.
Claim 28
Dependent Claim
13 words
The method according to claim 25 wherein the 5′-nuclease cleavable probe comprises FAM.
Claim 29
Dependent Claim
13 words
The method according to claim 25 wherein the 5′-nuclease cleavable probe comprises VIC.
Claim 30
Dependent Claim
15 words
The method according to claim 21 wherein the detector probe comprises peptide nucleic acid (PNA).
Claim 31
Dependent Claim
12 words
The method according to claim 28 wherein the PNA probe comprises FAM.
Claim 32
Dependent Claim
12 words
The method according to claim 28 wherein the PNA probe comprises VIC.
Claim 33
Dependent Claim
15 words
The method according to claim 21 wherein the detector probe comprises locked nucleic acid (LNA).
Claim 34
Dependent Claim
14 words
The method according to claim 21 wherein the detector probe comprises a universal base.
Claim 35
Dependent Claim
17 words
The method according to claim 21 the extending is a reverse transcription reaction comprising a reverse transcriptase.
Claim 36
Dependent Claim
16 words
The method according to claim 21 wherein the stem of the linker probe comprises 12-16 base-pairs.
Claim 37
Dependent Claim
16 words
The method according to claim 34 wherein the stem of the linker probe comprises 14 base-pairs.
Claim 38
Dependent Claim
19 words
The method according to claim 21 wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides.
Claim 39
Dependent Claim
16 words
The method according to claim 21 wherein the loop further comprises a universal reverse primer portion.
Claim 40
Dependent Claim
12 words
The method according to claim 21 wherein the loop comprises 14-18 nucleotides.
Claim 41
Dependent Claim
12 words
The method according to claim 38 wherein the loop comprises 16 nucleotides.
Claim 42
Dependent Claim
16 words
The method according to claim 21 wherein the Tm of the detector probe is 63-69 C.
Claim 43
Independent Claim
227 words
A method for detecting a miRNA molecule comprising; hybridizing the miRNA molecule and a linker probe, wherein the miRNA molecule comprises a 3′ end region, 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 molecule; 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 amplification comprises a polymerase chain reaction, wherein the amplification comprises: a forward primer that hybridizes to the target polynucleotide or a complementary sequence to the target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and the detector probe further hybridizes to a nucleotide of the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the miRNA in the amplification product, with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the miRNA molecule.
Claim 44
Independent Claim
227 words
A method for detecting a miRNA molecule comprising; hybridizing the miRNA molecule and a linker probe, wherein the miRNA molecule comprises a 3′ end region, 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 molecule; 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 amplification comprises a polymerase chain reaction, wherein the amplification comprises: a forward primer that hybridizes to the target polynucleotide or a complementary sequence to the target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the detector probe hybridizes to a nucleotide of the linker probe stem in the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and the detector probe further hybridizes to a nucleotide of the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the miRNA in the amplification product, with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the miRNA molecule.
Claim 45
Dependent Claim
14 words
The method according to claim 41 wherein the miRNA is 18-25 ribonucleotides in length.
Claim 46
Dependent Claim
15 words
The method according to claim 41 wherein the detector probe is a 5′-nuclease cleavable probe.
Claim 47
Dependent Claim
13 words
The method according to claim 43 wherein the 5′-nuclease cleavable probe comprises FAM.
Claim 48
Dependent Claim
13 words
The method according to claim 43 wherein the 5′-nuclease cleavable probe comprises VIC.
Claim 49
Dependent Claim
15 words
The method according to claim 41 wherein the detector probe comprises peptide nucleic acid (PNA).
Claim 50
Dependent Claim
12 words
The method according to claim 46 wherein the PNA probe comprises FAM.
Claim 51
Dependent Claim
12 words
The method according to claim 46 wherein the PNA probe comprises VIC.
Claim 52
Dependent Claim
15 words
The method according to claim 41 wherein the detector probe comprises locked nucleic acid (LNA).
Claim 53
Dependent Claim
14 words
The method according to claim 41 wherein the detector probe comprises a universal base.
Claim 54
Dependent Claim
18 words
The method according to claim 41 wherein the extending is a reverse transcription reaction comprising a reverse transcriptase.
Claim 55
Dependent Claim
16 words
The method according to claim 41 wherein the stem of the linker probe comprises 12-16 base-pairs.
Claim 56
Dependent Claim
16 words
The method according to claim 52 wherein the stem of the linker probe comprises 14 base-pairs.
Claim 57
Dependent Claim
19 words
The method according to claim 41 wherein the 3′ target specific portion of the linker probe comprises 5-8 nucleotides.
Claim 58
Dependent Claim
16 words
The method according to claim 41 wherein the loop further comprises a universal reverse primer portion.
Claim 59
Dependent Claim
12 words
The method according to claim 41 wherein the loop comprises 14-18 nucleotides.
Claim 60
Dependent Claim
12 words
The method according to claim 56 wherein the loop comprises 16 nucleotides.
Claim 61
Dependent Claim
16 words
The method according to claim 41 wherein the Tm of the detector probe is 63-69 C.
Claim 62
Independent Claim
446 words
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 a 3′ end region of the first miRNA, and wherein the 3′ target-specific portion of the second linker probe base pairs with a 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 vessel to form a first amplification reaction product, and a second amplification reaction vessel to form a second amplification reaction product, wherein within the first amplification reaction vessel is a first polymerase chain reaction vessel and within the second amplification reaction vessel is a second polymerase chain reaction vessel, wherein the first polymerase chain reaction vessel comprises a forward primer that hybridizes to the first miRNA or a complementary sequence to the first miRNA, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the second polymerase chain reaction vessel comprises a forward primer that hybridizes to the second miRNA or a complementary sequence to the second miRNA, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein a primer in the first amplification reaction vessel hybridizes to the first miRNA, or a complementary sequence to the first miRNA, and not the second miRNA, and a primer in the second amplification reaction vessel hybridizes to the second miRNA, or a complementary sequence to the first miRNA, and not the first miRNA, wherein a first detector probe in the first amplification reaction vessel differs from a second detector probe in the second amplification reaction vessel, with the proviso that the first linker probe is a different molecule from the first detector probe and the second linker probe is a different molecule from the second detector probe; wherein the first detector probe hybridizes to a nucleotide of the first linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the first linker probe stem in the first amplification product, wherein the second detector probe hybridizes to a nucleotide of the second linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the second linker probe stem in the amplification product; and, detecting the two different miRNAs.
Claim 63
Independent Claim
446 words
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 a 3′ end region of the first miRNA, and wherein the 3′ target-specific portion of the second linker probe base pairs with a 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 vessel to form a first amplification reaction product, and a second amplification reaction vessel to form a second amplification reaction product, wherein within the first amplification reaction vessel is a first polymerase chain reaction vessel and within the second amplification reaction vessel is a second polymerase chain reaction vessel, wherein the first polymerase chain reaction vessel comprises a forward primer that hybridizes to the first miRNA or a complementary sequence to the first miRNA, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the second polymerase chain reaction vessel comprises a forward primer that hybridizes to the second miRNA or a complementary sequence to the second miRNA, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein a primer in the first amplification reaction vessel hybridizes to the first miRNA, or a complementary sequence to the first miRNA, and not the second miRNA, and a primer in the second amplification reaction vessel hybridizes to the second miRNA, or a complementary sequence to the first miRNA, and not the first miRNA, wherein a first detector probe in the first amplification reaction vessel differs from a second detector probe in the second amplification reaction vessel, with the proviso that the first linker probe is a different molecule from the first detector probe and the second linker probe is a different molecule from the second detector probe; wherein the first detector probe hybridizes to a nucleotide of the first linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the first linker probe stem in the first amplification product, wherein the second detector probe hybridizes to a nucleotide of the second linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the second linker probe stem in the amplification product; and, detecting the two different miRNAs.
Claim 64
Dependent Claim
33 words
The method according to claim 59 wherein the reverse primer in the first polymerase chain reaction vessel and the reverse primer in the second polymerase chain reaction vessel are a universal reverse primer.
Claim 65
Dependent Claim
19 words
The method according to claim 59 wherein the first miRNA and/or the second miRNA is 18-25 ribonucleotides in length.
Claim 66
Dependent Claim
21 words
The method according to claim 59 wherein the first detector probe and/or the second detector probe is a 5′-nuclease cleavable probe.
Claim 67
Dependent Claim
18 words
The method according to claim 62 wherein the first detector probe and/or the second detector probe comprises FAM.
Claim 68
Dependent Claim
18 words
The method according to claim 62 wherein the first detector probe and/or the second detector probe comprises VIC.
Claim 69
Dependent Claim
21 words
The method according to claim 59 wherein the first detector probe and/or the second detector probe comprises peptide nucleic acid (PNA).
Claim 70
Dependent Claim
17 words
The method according to claim 65 wherein first detector probe and/or the second detector probe comprises FAM.
Claim 71
Dependent Claim
18 words
The method according to claim 65 wherein the first detector probe and/or the second detector probe comprises VIC.
Claim 72
Dependent Claim
21 words
The method according to claim 59 wherein the first detector probe and/or the second detector probe comprises locked nucleic acid (LNA).
Claim 73
Dependent Claim
20 words
The method according to claim 59 wherein the first detector probe and/or the second detector probe comprises a universal base.
Claim 74
Dependent Claim
18 words
The method according to claim 59 wherein the extending is a reverse transcription reaction comprising a reverse transcriptase.
Claim 75
Dependent Claim
22 words
The method according to claim 59 wherein the stem of the first linker probe and/or the second linker probe comprises 12-16 base-pairs.
Claim 76
Dependent Claim
22 words
The method according to claim 71 wherein the stem of the first linker probe and/or the second linker probe comprises 14 base-pairs.
Claim 77
Dependent Claim
25 words
The method according to claim 59 wherein the 3′ target specific portion of the first linker probe and/or the second linker probe comprises 5-8 nucleotides.
Claim 78
Dependent Claim
26 words
The method according to claim 59 wherein the loop of the first linker probe and/or the second linker probe further comprises a universal reverse primer portion.
Claim 79
Dependent Claim
22 words
The method according to claim 59 wherein the loop of the first linker probe and/or the second linker probe comprises 14-18 nucleotides.
Claim 80
Dependent Claim
22 words
The method according to claim 75 wherein the loop of the first linker probe and/or the second linker probe comprises 16 nucleotides.
Claim 81
Dependent Claim
22 words
The method according to claim 59 wherein the Tm of the first detector probe and/or the second detector probe is 63-69 C.
Claim 82
Independent Claim
419 words
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 a 3′ end region of the first target polynucleotide, wherein the 3′ target-specific portion of the second linker probe base pairs with a 3′ end region of the second target polynucleotide, wherein the first target polynucleotide is 18 to 25 nucleotides in length, and wherein the second target polynucleotide is 18 to 25 nucleotides in length; 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 vessel to form a first amplification reaction product in the presence of a first detector probe and a second amplification reaction vessel to form a second amplification reaction product in the presence of a second detector probe, with the proviso that the first linker probe is a different molecule from the first detector probe, and the second liner probe is a different molecule from the second detector probe, wherein within the first amplification reaction vessel is a first polymerase chain reaction and within the second amplification reaction vessel is a second polymerase chain reaction; wherein the first polymerase chain reaction comprises a forward primer that hybridizes to the first target polynucleotide or a complementary sequence to the first target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the second polymerase chain reaction comprises a forward primer that hybridizes to the second target polynucleotide or a complementary sequence to the second target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the second linker probe; and, detecting the first target polynucleotide and the second target polynucleotide, wherein the first detector probe hybridizes to a nucleotide of the first linker probe in the first amplification product or hybridizes to a nucleotide of a complementary sequence to the first linker probe in the first amplification product, and/or the second detector probe hybridizes to a nucleotide of the second linker probe in the second amplification product or hybridizes to a nucleotide of a complementary sequence to the second linker probe in the second amplification product.
Claim 83
Independent Claim
419 words
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 a 3′ end region of the first target polynucleotide, wherein the 3′ target-specific portion of the second linker probe base pairs with a 3′ end region of the second target polynucleotide, wherein the first target polynucleotide is 18 to 25 nucleotides in length, and wherein the second target polynucleotide is 18 to 25 nucleotides in length; 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 vessel to form a first amplification reaction product in the presence of a first detector probe and a second amplification reaction vessel to form a second amplification reaction product in the presence of a second detector probe, with the proviso that the first linker probe is a different molecule from the first detector probe, and the second liner probe is a different molecule from the second detector probe, wherein within the first amplification reaction vessel is a first polymerase chain reaction and within the second amplification reaction vessel is a second polymerase chain reaction; wherein the first polymerase chain reaction comprises a forward primer that hybridizes to the first target polynucleotide or a complementary sequence to the first target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the linker probe, wherein the second polymerase chain reaction comprises a forward primer that hybridizes to the second target polynucleotide or a complementary sequence to the second target polynucleotide, and a reverse primer that hybridizes to the linker probe or a complementary sequence to the second linker probe; and, detecting the first target polynucleotide and the second target polynucleotide, wherein the first detector probe hybridizes to a nucleotide of the first linker probe in the first amplification product or hybridizes to a nucleotide of a complementary sequence to the first linker probe in the first amplification product, and/or the second detector probe hybridizes to a nucleotide of the second linker probe in the second amplification product or hybridizes to a nucleotide of a complementary sequence to the second linker probe in the second amplification product.
Claim 84
Dependent Claim
31 words
The method according to claim 78 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.
Claim 85
Dependent Claim
15 words
The method according to claim 78 wherein the target polynucleotide is a micro RNA (miRNA).
Claim 86
Dependent Claim
28 words
The method according to claim 78 wherein the first amplification reaction vessel comprises a first detector probe and/or the second amplification reaction vessel comprises a second detector probe.
Claim 87
Dependent Claim
86 words
The method according to claim 81 wherein the first detector probe hybridizes to a nucleotide of the first linker probe stem of the first amplification product or hybridizes to a nucleotide of a complementary sequence to the first linker probe stem in the first amplification product, and/or the second detector probe hybridizes to a nucleotide of the second linker probe stem in the second amplification product or hybridizes to a nucleotide of a complementary sequence to the second linker probe stem in the second amplification product.
Claim 88
Dependent Claim
102 words
The method according to claim 81 wherein the first detector probe hybridizes to a nucleotide of the 3′ end region of the first target polynucleotide in the first amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the first target polynucleotide in the first amplification product, and/or the second detector probe hybridizes to a nucleotide of the 3′ end region of the second target polynucleotide in the second amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the second target polynucleotide in the second amplification product.
Claim 89
Dependent Claim
118 words
The method according to claim 81 wherein the first detector probe hybridizes to a nucleotide of a region upstream from the 3′ end region of the first target polynucleotide in the first amplification product or hybridizes to a nucleotide of a complementary sequence to a region upstream from the 3′ end region of the first target polynucleotide in the first amplification product, and/or the second detector probe hybridizes to a nucleotide of a region upstream from the 3′ end region of the second target polynucleotide in the second amplification product or hybridizes to a nucleotide of a complementary sequence to a region upstream from the 3′ end region of the second target polynucleotide in the second amplification product.
Claim 90
Dependent Claim
21 words
The method according to claim 78 wherein the first target polynucleotide and/or the second target polynucleotide is 18-25 ribonucleotides in length.
Claim 91
Dependent Claim
20 words
The method according to claim 81 wherein the first detector probe and/or second detector probe is a 5′-nuclease cleavable probe.
Claim 92
Dependent Claim
17 words
The method according to claim 86 wherein the first detector probe and/or second detector probe comprises FAM.
Claim 93
Dependent Claim
17 words
The method according to claim 86 wherein the first detector probe and/or second detector probe comprises VIC.
Claim 94
Dependent Claim
20 words
The method according to claim 81 wherein the first detector probe and/or second detector probe comprises peptide nucleic acid (PNA).
Claim 95
Dependent Claim
16 words
The method according to claim 89 wherein first detector probe and/or second detector probe comprises FAM.
Claim 96
Dependent Claim
17 words
The method according to claim 89 wherein the first detector probe and/or second detector probe comprises VIC.
Claim 97
Dependent Claim
21 words
The method according to claim 81 wherein the first detector probe and/or the second detector probe comprises locked nucleic acid (LNA).
Claim 98
Dependent Claim
20 words
The method according to claim 81 wherein the first detector probe and/or the second detector probe comprises a universal base.
Claim 99
Dependent Claim
18 words
The method according to claim 78 wherein the extending is a reverse transcription reaction comprising a reverse transcriptase.
Claim 100
Dependent Claim
22 words
The method according to claim 78 wherein the stem of the first linker probe and/or the second linker probe comprises 12-16 base-pairs.
Claim 101
Dependent Claim
22 words
The method according to claim 95 wherein the stem of the first linker probe and/or the second linker probe comprises 14 base-pairs.
Claim 102
Dependent Claim
25 words
The method according to claim 78 wherein the 3′ target specific portion of the first linker probe and/or the second linker probe comprises 5-8 nucleotides.
Claim 103
Dependent Claim
25 words
The method according to claim 78 wherein the loop of the first linker probe and/or the second linker probe comprises a universal reverse primer portion.
Claim 104
Dependent Claim
22 words
The method according to claim 78 wherein the loop of the first linker probe and/or the second linker probe comprises 14-18 nucleotides.
Claim 105
Dependent Claim
22 words
The method according to claim 99 wherein the loop of the first linker probe and/or the second linker probe comprises 16 nucleotides.
Claim 106
Dependent Claim
21 words
The method according to claim 81 wherein the Tm of the first detector probe and/or second detector probe is 63-69 C.
Claim 107
Independent Claim
176 words
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 a 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 simultaneously hybridizes to both a) a nucleotide of the linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and b) a nucleotide of the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the miRNA in the amplification product, with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the miRNA molecule.
Claim 108
Independent Claim
176 words
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 a 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 simultaneously hybridizes to both a) a nucleotide of the linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and b) a nucleotide of the 3′ end region of the miRNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the miRNA in the amplification product, with the proviso that the linker probe is a different molecule from the detector probe; and, detecting the miRNA molecule.
Claim 109
Dependent Claim
34 words
The method according to claim 102 , 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.
Claim 110
Independent Claim
159 words
A method for detecting a RNA, wherein the RNA is 18-25 ribonucleotides in length, comprising; hybridizing the RNA 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 a 3′ end region of the RNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in a polymerase chain reaction comprising a detector probe to form an amplification product, wherein the detector probe simultaneously hybridizes to both a) a nucleotide of the linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and b) a nucleotide of the 3′ end region of the RNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the RNA in the amplification product; and, detecting the RNA.
Claim 111
Independent Claim
159 words
A method for detecting a RNA, wherein the RNA is 18-25 ribonucleotides in length, comprising; hybridizing the RNA 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 a 3′ end region of the RNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in a polymerase chain reaction comprising a detector probe to form an amplification product, wherein the detector probe simultaneously hybridizes to both a) a nucleotide of the linker probe stem of the amplification product or hybridizes to a nucleotide of a complementary sequence to the linker probe stem in the amplification product, and b) a nucleotide of the 3′ end region of the RNA in the amplification product or hybridizes to a nucleotide of a complementary sequence to the 3′ end region of the RNA in the amplification product; and, detecting the RNA.
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