Chemical capping for template switching
AssigneeNew England Biolabs, Inc.
InventorsIvan R. Correa, JR., Shengxi Guan, Madalee G. Wulf, Nan Dai, Sean Maguire
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Abstract
Provided herein is a method for chemically capping polynucleotides having a 5′ monophosphate. In some embodiments the method may comprise: combining an activated nucleoside 5′ mono- or poly-phosphate with a population of polynucleotides that comprises polynucleotides having a 5′ monophosphate, to produce a reaction mix; and incubating the reaction mix to produce reaction products that comprise a polynucleotide and a 5′ nucleoside cap, linked by a 5′ to 5′ polyphosphate linkage. The chemical capping method described herein can be incorporated into a variety of cDNA synthesis methods.
Claims24
-34. (canceled)
A method for synthesizing cDNA from a population of single-strand polynucleotides, comprising:
- (a) combining an activated nucleoside 5′ mono- or polyphosphate comprising a phosphorodichloridate, phosphoramidate, phosphodiester, phosphotriester, 5′-H-phosphonate, P(III)-P(V) mixed anhydride, or phosphite triesters, with the population of single-strand polynucleotides, wherein each polynucleotide in the population comprises a 5′ monophosphate, to produce a reaction mix;
- (b) incubating the reaction mix to chemically cap the population of polynucleotides and produce 5′ capped polynucleotides that each comprise a single-strand polynucleotide linked to a 5′ nucleoside cap by a 5′ to 5′ polyphosphate linkage;
- (c) contacting the 5′ capped polynucleotides with a template switching oligonucleotide (TSO) and a reverse transcriptase (RT);
- and
- (d) synthesizing via template switching cDNA complementary to the single-strand polynucleotides and the TSO.
A kit comprising a nucleoside 5′-phosphoroimidazolide, a capping buffer pH 5-pH 6.5, a reverse transcriptase, and a template switching oligonucleotide (TSO), in one or more different storage containers.
A kit comprising a plurality of modules, each module being in one or more containers, wherein:
- a first module is a capping module comprising reagents for chemically capping a population of single-strand polynucleotides that comprise a 5′ monophosphate, wherein the first module reagents comprise an activated nucleoside 5′ mono- or polyphosphate comprising a phosphorodichloridate, phosphoramidate, phosphodiester, phosphotriester, 5′-H-phosphonate, P(III)-P(V) mixed anhydride, or phosphite triesters;
- and
- a second module comprises a cDNA synthesis and amplification module, wherein the second module includes a template switching oligonucleotide (TSO) and optionally further includes a 3′ splint adaptor.
Dependencies are inferred from the claim text.