A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

A method for sequencing a nucleic acid strand, comprising the steps of: providing a solution containing truncated strands having lengths different from one another terminating with a respective dideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP; functionalizing first masses by a donor molecule and second masses by an acceptor molecule such as to generate a light emission when they come into mutual contact; coupling a first mass to a first end of each truncated strand; coupling the second masses to a respective terminal dideoxynucleotide of each strand; applying an AC electrical field having variable frequencies that are such as to generate, on each second mass, a net movement directed towards the first mass; acquiring a plurality of light radiations for each frequency value; and associating each light radiation acquired to a respective dideoxynucleotide and, thus, to a respective nucleotide base.

A method for sequencing a nucleic acid strand, comprising the steps of: providing a solution containing truncated strands having lengths different from one another terminating with a respective dideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP; functionalizing first masses by a donor molecule and second masses by an acceptor molecule such as to generate a light emission when they come into mutual contact; coupling a first mass to a first end of each truncated strand; coupling the second masses to a respective terminal dideoxynucleotide of each strand; applying an AC electrical field having variable frequencies that are such as to generate, on each second mass, a net movement directed towards the first mass; acquiring a plurality of light radiations for each frequency value; and associating each light radiation acquired to a respective dideoxynucleotide and, thus, to a respective nucleotide base.

Provided herein are products and processes for detecting the presence or absence of multiple target nucleic acids. Certain methods include amplifying the target nucleic acids, or portion thereof; extending oligonucleotides that specifically hybridize to the amplicons, where the extended oligonucleotides include a capture agent; capturing the extended oligonucleotides to a solid phase via the capture agent; releasing the extended oligonucleotide by competition with a competitor; detecting the extended oligonucleotide, and thereby determining the presence or absence of each target nucleic acid by the presence or absence of the extended oligonucleotide.

Provided herein are products and processes for detecting the presence or absence of multiple target nucleic acids. Certain methods include amplifying the target nucleic acids, or portion thereof; extending oligonucleotides that specifically hybridize to the amplicons, where the extended oligonucleotides include a capture agent; capturing the extended oligonucleotides to a solid phase via the capture agent; releasing the extended oligonucleotide by competition with a competitor; detecting the extended oligonucleotide, and thereby determining the presence or absence of each target nucleic acid by the presence or absence of the extended oligonucleotide.

A method of sample analysis is provided. In certain embodiments, the method may comprise: contacting a nucleic acid sample with a first primer and a second primer under PCR conditions to produce a double stranded product, wherein the second primer comprises a first label and is 5′ blocked; b) contacting the double stranded product with an exonuclease to degrade one strand of the double-stranded product to produce a single stranded product; c) contacting the single stranded product with a third primer under primer extension conditions, wherein the third primer comprises a second label; and d) detecting the first and second labels of the partial duplex. A kit for practicing the method is also provided.

A method of sample analysis is provided. In certain embodiments, the method may comprise: contacting a nucleic acid sample with a first primer and a second primer under PCR conditions to produce a double stranded product, wherein the second primer comprises a first label and is 5′ blocked; b) contacting the double stranded product with an exonuclease to degrade one strand of the double-stranded product to produce a single stranded product; c) contacting the single stranded product with a third primer under primer extension conditions, wherein the third primer comprises a second label; and d) detecting the first and second labels of the partial duplex. A kit for practicing the method is also provided.

The present disclosure relates to compositions that comprise a fluorescent and/or MS-active nucleic acid probe that comprises (a) a nucleic-acid-based moiety and (b) a fluorescent moiety comprising a fluorophore, an MS-active moiety, or both a fluorescent moiety and an MS-active moiety, linked to the nucleic-acid-based moiety, among other aspects.

The present disclosure relates to compositions that comprise a fluorescent and/or MS-active nucleic acid probe that comprises (a) a nucleic-acid-based moiety and (b) a fluorescent moiety comprising a fluorophore, an MS-active moiety, or both a fluorescent moiety and an MS-active moiety, linked to the nucleic-acid-based moiety, among other aspects.