COMPOSITIONS, METHODS, AND KITS FOR DETECTING RIBONUCLEIC ACID
First Claim
1. A method for detecting a RNA molecule in a sample, comprising:
- combining the sample with at least one first adaptor, at least one second adaptor, and a polypeptide comprising double-strand specific RNA ligase activity to form a ligation reaction composition in which the at least one first adaptor and the at least one second adaptor are ligated to the RNA molecule of the sample to form a ligated product in the same ligation reaction composition,wherein the at least one first adaptor comprises;
a first oligonucleotide having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3′
-end, anda second oligonucleotide comprising a nucleotide sequence substantially complementary to the first oligonucleotide and further comprising a single-stranded 5′
portion of 1 to 8 nucleotides when the first oligonucleotide and the second oligonucleotide are duplexed,wherein the at least one second adaptor comprises;
a third oligonucleotide having a length of 10 to 60 nucleotides and comprising a 5′
phosphate group, anda fourth oligonucleotide comprising a nucleotide sequence substantially complementary to the third oligonucleotide and further comprising a single-stranded 3′
portion of 1 to 8 nucleotides when the third oligonucleotide and the fourth oligonucleotide are duplexed,wherein the single-stranded portions independently have a degenerate nucleotide sequence, or a sequence that is complementary to a portion of the RNA molecule,wherein the first and third oligonucleotides have a different nucleotide sequence;
wherein the RNA molecule hybridizes with the single-stranded portion of the at least one first adaptor and the single-stranded portion of the at least one second adaptor; and
detecting the RNA molecule of the ligated product or a surrogate thereof.
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Accused Products
Abstract
Compositions, methods, and kits for detecting one or more species of RNA molecules are disclosed. In one embodiment, a first adaptor and a second adaptor are ligated to the RNA molecule using a polypeptide comprising double-strand specific RNA ligase activity, without an intervening purification step. The ligated product is reverse transcribed, then at least some of the ribonucleosides in the reverse transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, the sequence of at least part of at least one species of amplified product is determined and at least part of the corresponding RNA molecule is determined. In some embodiments, at least some of the amplified product species are detected, directly or indirectly, allowing the presence and/or quantity of the RNA molecule of interest to be determined.
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Citations
38 Claims
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1. A method for detecting a RNA molecule in a sample, comprising:
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combining the sample with at least one first adaptor, at least one second adaptor, and a polypeptide comprising double-strand specific RNA ligase activity to form a ligation reaction composition in which the at least one first adaptor and the at least one second adaptor are ligated to the RNA molecule of the sample to form a ligated product in the same ligation reaction composition, wherein the at least one first adaptor comprises; a first oligonucleotide having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3′
-end, anda second oligonucleotide comprising a nucleotide sequence substantially complementary to the first oligonucleotide and further comprising a single-stranded 5′
portion of 1 to 8 nucleotides when the first oligonucleotide and the second oligonucleotide are duplexed,wherein the at least one second adaptor comprises; a third oligonucleotide having a length of 10 to 60 nucleotides and comprising a 5′
phosphate group, anda fourth oligonucleotide comprising a nucleotide sequence substantially complementary to the third oligonucleotide and further comprising a single-stranded 3′
portion of 1 to 8 nucleotides when the third oligonucleotide and the fourth oligonucleotide are duplexed,wherein the single-stranded portions independently have a degenerate nucleotide sequence, or a sequence that is complementary to a portion of the RNA molecule, wherein the first and third oligonucleotides have a different nucleotide sequence; wherein the RNA molecule hybridizes with the single-stranded portion of the at least one first adaptor and the single-stranded portion of the at least one second adaptor; and detecting the RNA molecule of the ligated product or a surrogate thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17-30. -30. (canceled)
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31. A method for detecting an RNA molecule, comprising:
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combining the RNA molecule with at least one first adaptor, at least one second adaptor, and a double-strand specific RNA ligase to form a ligation reaction composition, wherein the at least one first adaptor comprises a first oligonucleotide comprising at least two ribonucleosides on the 3′
-end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5′
phosphate group and a fourth oligonucleotide that comprises a single-stranded portion when the third oligonucleotide and the fourth oligonucleotide are hybridized together,ligating the at least one first adaptor and the at least one second adaptor to the RNA molecule to form a ligated product, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the same ligation reaction composition, combining the ligated product with an RNA-directed DNA polymerase, reverse transcribing the ligated product to form a reverse transcribed product, digesting at least some of the ribonucleosides from the reverse transcribed product with a ribonuclease H(RNase H) to form an amplification template, combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase to form an amplification reaction composition, cycling the amplification reaction composition to form an amplified product, wherein the amplification reaction composition further comprises a reporter probe, a nucleic acid dye, or a reporter probe and a nucleic acid dye, and detecting the amplified product, thereby detecting the RNA molecule.
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32. A method for generating an RNA library comprising,
combining a multiplicity of different RNA molecules with a multiplicity of first adaptor species, a multiplicity of second adaptor species, and a double-strand specific RNA ligase to form a ligation reaction composition, wherein the at least one first adaptor comprises a first oligonucleotide comprising at least two ribonucleosides on the 3′ - -end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5′
phosphate group and a fourth oligonucleotide that comprises a single-stranded portion when the third oligonucleotide and the fourth oligonucleotide are hybridized together,ligating the at least one first adaptor and the at least one second adaptor to the RNA molecule to form a multiplicity of different ligated product species, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the same ligation reaction composition, combining the multiplicity of ligated product species with an RNA-directed DNA polymerase, reverse transcribing at least some of the multiplicity of ligated product species to form a multiplicity of reverse transcribed product species, digesting at least some of the ribonucleosides from at least some of the multiplicity of reverse transcribed products with a ribonuclease H(RNase H) to form a multiplicity of amplification template species, combining the multiplicity of amplification template species with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase to form an amplification reaction composition, cycling the amplification reaction composition to form a library comprising a multiplicity of amplified product species, wherein at least some of the amplified product species comprise an identification sequence that is common to at least some of the other amplified product species in the library. - View Dependent Claims (33)
- -end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5′
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34. A kit comprising:
- a plurality of first adaptor species, wherein each first adaptor species comprises a different degenerate sequence, a plurality of second adaptor species, wherein each second adaptor species comprises a different degenerate sequence, a ligase of the Rnl2 family, a RNA-directed DNA polymerase, a plurality of different first primer species, a DNA-directed DNA polymerase and RNase H (EC 3.1.26.4).
- View Dependent Claims (35)
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36. A kit comprising:
- a plurality of first adaptor species, wherein at least some of the first adaptor species comprise a degenerate sequence, a plurality of second adaptor species, wherein at least some of the second adaptor species comprise a degenerate sequence, a polypeptide comprising double-strand specific RNA ligase activity, a DNA polymerase, at least one primer species, and ribonuclease H.
- 37. The kit of claim 37, wherein the DNA polymerase comprises an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase.
Specification