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.
Claims34
A method for chemically capping a population of polynucleotides having a 5′ monophosphate, comprising:
- (a) combining an activated nucleoside 5′ mono-phosphate or an activated nucleoside 5′ poly-phosphate with the population of polynucleotides, to produce a reaction mix;
- and
- (b) incubating the reaction mix to produce reaction products that each comprise a polynucleotide linked to a 5′ nucleoside cap by a 5′ to 5′ polyphosphate linkage.
A method for synthesizing a cDNA from a single strand polynucleotide, comprising the steps of:
- (a) combining an activated nucleoside 5′ mono- or poly-phosphate with a population of polynucleotides having a 5′ monophosphate, to produce a reaction mix;
- (b) incubating the reaction mix to produce reaction products that each comprise a polynucleotide and a 5′ nucleoside cap, linked by a 5′ to 5′ polyphosphate linkage;
- and
- (c) reverse transcribing the products of step (c) in the presence of template switching oligonucleotide (TSO) to produce cDNA that comprises the complement of the TSO at the cDNA 3′ end.
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