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US 2024/0182992 A1Published application
Methods and compositions related to cooperative primers and reverse transcription
AssigneePLC Diagnostics, Inc.
InventorKenneth K.C. BRAMWELL
Life of this patent
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Abstract
Disclosed are compositions, methods, and kits relating to amplifying and detecting RNA using Co-Primers and conditionally active reverse transcription primers. The conditionally active reverse transcriptase primers work in concert with dual Co-Primer assays to improve sensitivity toward RNA targets without detracting from the improved amplification specificity of the associated Co-Primers.
Claims25
A method of transcribing RNA into cDNA, the method comprising:
- a. providing a target RNA sequence;
- b. providing a cooperative nucleic acid primer (“co-primer”), wherein said co-primer comprises:
- i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of the target RNA, and wherein the first nucleic acid sequence is extendable on the 3′ end;
- ii. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target RNA, such that in the presence of the target RNA, the second nucleic acid sequence hybridizes to the target RNA downstream from the 3′ end of the first nucleic acid sequence;
- iii. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target RNA at the same time;
- c. providing a conditionally active reverse transcriptase primer, wherein said conditionally active reverse transcriptase primer has a melting temperature between about 45° C. and about 55° C.;
- d. providing reverse transcriptase;
- and
- e. providing conditions that allow for transcription of the RNA target into cDNA;
- thereby transcribing RNA into cDNA.
7 more elements
A kit comprising:
- a. a cooperative nucleic acid primer (“co-primer”), wherein said co-primer comprises:
- i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of the target RNA, and wherein the first nucleic acid sequence is extendable on the 3′ end;
- ii. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target RNA, such that in the presence of the target RNA, the second nucleic acid sequence hybridizes to the target RNA downstream from the 3′ end of the first nucleic acid sequence;
- iii. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target RNA at the same time;
- b. a conditionally active reverse transcriptase primer, wherein said conditionally active reverse transcriptase primer has a melting temperature between about 45° C. and about 55° C.
2 more elements
A method of detecting RNA, the method comprising:
- a. providing a target RNA sequence;
- b. providing a cooperative nucleic acid primer (“co-primer”), wherein said co-primer comprises:
- i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of the target RNA, and wherein the first nucleic acid sequence is extendable on the 3′ end;
- ii. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target RNA, such that in the presence of the target RNA, the second nucleic acid sequence hybridizes to the target RNA downstream from the 3′ end of the first nucleic acid sequence;
- iii. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target RNA at the same time;
- c. providing a conditionally active reverse transcriptase primer, wherein said conditionally active reverse transcriptase primer has a melting temperature between about 45° C. and about 55° C.;
- d. providing reverse transcriptase;
- e. providing conditions that allow for transcription of the RNA target into cDNA, thereby producing cDNA;
- f. amplifying the cDNA product of step e);
- and
- g. detecting the amplified cDNA produced in step f.
8 more elements
The method of step 11, wherein before said amplification step, appropriate reagents and conditions for amplification are provided.
Dependencies are inferred from the claim text.