Amplification and detection of ribonucleic acids
First Claim
1. A composition, comprising:
- a single reaction mixture containinga plurality of RNA molecules,a plurality of a first double-stranded nucleic acid adaptor,a plurality of a second double-stranded nucleic acid adaptor, anda double-strand specific RNA ligase,wherein a first strand of the first double-stranded nucleic acid adaptor of the plurality includes at least two ribonucleosides at the 3′
end, and a second strand of the first double-stranded nucleic acid adaptor of the plurality include a first single-stranded portion at the 5′
end, andwherein a first strand of the second double-stranded nucleic acid adaptor of the plurality includes a terminal 5′
phosphate group, and a second strand of the second double-stranded nucleic acid adaptor of the plurality include a second single-stranded portion at the 3′
end, andwherein the first single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a first region of a first RNA molecule in the plurality of RNA molecules, andwherein the second single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a second region of the first RNA molecule in the plurality of RNA molecules.
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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.
25 Citations
20 Claims
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1. A composition, comprising:
- a single reaction mixture containing
a plurality of RNA molecules, a plurality of a first double-stranded nucleic acid adaptor, a plurality of a second double-stranded nucleic acid adaptor, and a double-strand specific RNA ligase, wherein a first strand of the first double-stranded nucleic acid adaptor of the plurality includes at least two ribonucleosides at the 3′
end, and a second strand of the first double-stranded nucleic acid adaptor of the plurality include a first single-stranded portion at the 5′
end, andwherein a first strand of the second double-stranded nucleic acid adaptor of the plurality includes a terminal 5′
phosphate group, and a second strand of the second double-stranded nucleic acid adaptor of the plurality include a second single-stranded portion at the 3′
end, andwherein the first single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a first region of a first RNA molecule in the plurality of RNA molecules, and wherein the second single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a second region of the first RNA molecule in the plurality of RNA molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
- a single reaction mixture containing
Specification
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- Resources
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Current AssigneeApplied Biosystems LLC (Thermo Fisher Scientific Incorporated)
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Original AssigneeApplied Biosystems LLC (Thermo Fisher Scientific Incorporated)
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InventorsKuersten, R. Scott
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Primary Examiner(s)Horlick, Kenneth R
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Application NumberUS15/803,609Publication NumberTime in Patent Office508 DaysField of SearchNoneUS Class CurrentCPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...C12Q 1/68 involving nucleic acidsC12Q 1/6806 Preparing nucleic acids for...C12Q 1/6855 Ligating adaptorsC12Q 2521/107 RNA dependent DNA polymeras...C12Q 2525/207 siRNA, miRNAC12Q 2565/125 Electrophoretic separationC12Q 2565/137 Chromatographic separation
