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US 12,698,498 B2Granted
Compositions and methods for constructing strand specific cDNA libraries
AssigneeThe Regents of the University of California
InventorsBrad Townsley, Michael F. Covington, Neelima Sinha
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Abstract
Provided herein are compositions, kits and methods for the production of strand-specific cDNA libraries. The compositions, kits and methods utilize properties of double stranded polynucleotides, such as RNA-cDNA duplexes to capture and incorporate a novel sequencing adapter. The methods are useful transcriptome profiling by massive parallel sequence, such as full-length RNA sequencing (RNA-Seq) and 3′ tag digital gene expression (DGE).
Claims13
A method of generating a strand-specific DNA molecule from double stranded nucleic acid, the method comprising:
- providing a double-stranded nucleic acid duplex comprising a first strand and a second strand;
- without removal of the first strand or the second strand, annealing a partially double stranded oligonucleotide 5′ adapter to the 3′ end of the first strand in the duplex, wherein the 5′ adapter comprises:
- (i) a first strand capturing oligonucleotide comprising at least 20 nucleotides and a 3′ overhang comprising about 6-12 consecutive random nucleotides that anneal to the 3′ end of the first strand;
- and
- (ii) a second strand blocking oligonucleotide comprising at least 20 nucleotides complementary to at least a portion of the first strand capturing oligonucleotide;
- and
- generating the strand specific DNA molecule by extending the first strand capturing oligonucleotide of the 5′ adapter using a DNA polymerase or a fragment thereof to generate a DNA strand complementary to the first strand.
4 more elements
Dependencies are inferred from the claim text.