There is provided a method of synthesizing a single-stranded nucleotide sequence, the method comprising adding a blocked nucleoside triphosphate to an initiator nucleotide sequence to incorporate a corresponding blocked nucleotide thereto in the presence of a polymerase, wherein the blocked nucleoside triphosphate has one of the general formulae (I), (II), (III), (IV), (V) and (VI).

There is provided a method of synthesizing a single-stranded nucleotide sequence, the method comprising adding a blocked nucleoside triphosphate to an initiator nucleotide sequence to incorporate a corresponding blocked nucleotide thereto in the presence of a polymerase, wherein the blocked nucleoside triphosphate has one of the general formulae (I), (II), (III), (IV), (V) and (VI).

Provided herein are compositions and methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for research, diagnostics, and treatment.

Provided herein are compositions and methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for research, diagnostics, and treatment.

There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.

There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

The present invention provides methods, antibodies and kits for detecting and/or quantifying expression of both non-nucleic acid molecules, such as proteins, and nucleic acid molecules in a single sample or single cell. The antibody is covalently conjugated to an oligonucleotide, such as a single-stranded DNA molecule, which comprises an identification tag and a blocking group, such as a ddNTP or an inverted dTTP, which prevents extension of the oligonucleotide by a polymerase. The method comprises the steps of reverse transcribing the conjugated oligonucleotide and the target nucleic acid simultaneously, amplifying both transcription production, and detecting the amplicons thereof. The method is also useful for detecting and/or quantifying the number of cells in a sample expressing a given non -nucleic acid molecule (e.g. protein).

The present invention provides methods, antibodies and kits for detecting and/or quantifying expression of both non-nucleic acid molecules, such as proteins, and nucleic acid molecules in a single sample or single cell. The antibody is covalently conjugated to an oligonucleotide, such as a single-stranded DNA molecule, which comprises an identification tag and a blocking group, such as a ddNTP or an inverted dTTP, which prevents extension of the oligonucleotide by a polymerase. The method comprises the steps of reverse transcribing the conjugated oligonucleotide and the target nucleic acid simultaneously, amplifying both transcription production, and detecting the amplicons thereof. The method is also useful for detecting and/or quantifying the number of cells in a sample expressing a given non -nucleic acid molecule (e.g. protein).