Compositions for cDNA Synthesis and Transcriptome Profiling and Methods of Use Thereof
AssigneeYale University
InventorsAnna Marie Pyle, Li-Tao Guo
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Abstract
The present invention provides optimized template switching oligonucleotides, methods, and kits for performing reverse transcription. The optimized template switching oligonucleotides include modifications of 5′ and 3′ ends to prevent the formation of concatemers and to enhance the specificity of reverse transcription.
Claims73
A template switching oligonucleotide (TSO) comprising:
- (i) a DNA nucleotide sequence, an RNA nucleotide sequence, or a hybrid DNA-RNA sequence;
- and
- (ii) at least one of a 3′ end modification and a 5′ end modification.
A template switching oligonucleotide (TSO) comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the TSO can anneal by base-pairing to non-templated nucleotides that have been added to the 5′-end of a target nucleic acid molecule during a non-templated addition by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3′ end modification and a 5′ end modification.
A reverse transcription (RT) primer comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the RT primer comprises a 5′ end modification.
A reverse transcription assay for of generating a cDNA molecule from an RNA template, the method comprising contacting an RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the TSO can anneal by base-pairing to non-templated nucleotides that have been added to the 5′-end of a target nucleic acid molecule during a non-templated addition by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3′ end modification and a 5′ end modification, a reverse transcription (RT) primer and a reverse transcriptase.
A kit for performing an assay for generating a cDNA molecule from an RNA template, the method comprising contacting an RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the TSO can anneal by base-pairing to non-templated nucleotides that have been added to the 5′-end of a target nucleic acid molecule during a non-templated addition by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3′ end modification and a 5′ end modification, a reverse transcription (RT) primer and a reverse transcriptase.
A method of reducing the concatemerization of a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase, wherein the TSO comprises:
- (i) a 3′ end modification;
- and/or
- (ii) a 5′ end modification.
A method of reducing the non-specific reverse transcription from a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase,
- wherein the TSO comprises:
- (i) a 3′ end modification;
- and/or
- (ii) a 5′ end modification.
A method of increasing yield of target polynucleotide sequences in a RNA-seq library, the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase,
- wherein the TSO comprises:
- (i) a 3′ end modification;
- and/or
- (ii) a 5′ end modification.
A method of increasing the specificity of an RNA-seq library, the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase,
- wherein the TSO comprises:
- (i) a 3′ end modification;
- and/or
- (ii) a 5′ end modification;
- wherein the RNA-seq library is prepared using template switching.
Dependencies are inferred from the claim text.