Methods for quenching a nuclease digestion of a target nucleic acid prior to downstream analysis of the target nucleic acid are disclosed herein. Particularly, methods for controlling the end point of a nuclease digestion prior to sequence analysis of a target nucleic acid is provided. Quenching of a nuclease digestion in the present disclosure employs at least one non-ionic or anionic denaturant combined with an optional reducing agent. The methods presented in this disclosure aids preserving the sample comprising the target nucleic acid or fragments thereof for long term storage and ensures that the effect of contaminating nucleases is eliminated during pretreatment step.

Methods for quenching a nuclease digestion of a target nucleic acid prior to downstream analysis of the target nucleic acid are disclosed herein. Particularly, methods for controlling the end point of a nuclease digestion prior to sequence analysis of a target nucleic acid is provided. Quenching of a nuclease digestion in the present disclosure employs at least one non-ionic or anionic denaturant combined with an optional reducing agent. The methods presented in this disclosure aids preserving the sample comprising the target nucleic acid or fragments thereof for long term storage and ensures that the effect of contaminating nucleases is eliminated during pretreatment step.

Nucleic acid sequencing methods and systems, the systems including nanochannel chip including: a nanochannel formed in an upper surface of the nanochannel chip and; a roof covering the nanochannel and comprising nanopores and a field enhancement structure; and a barrier disposed in the nanochannel. The method including: introducing a buffer solution including long-chain nucleic acids to the nanochannel chip; applying a voltage potential across the nanochannel chip to drive the nucleic acids through the nanochannel, towards the barrier, and to translocate the nucleic acids through nanopores adjacent to the barrier, such that bases of each of the nucleic acids pass through the field enhancement structure one base at a time and emerge onto an upper surface of the roof; detecting the Raman spectra of the bases of the nucleic acids as each base passes through the electromagnetic-field enhancement structure; and sequencing the nucleic acids based on the detected Raman spectra.

Nucleic acid sequencing methods and systems, the systems including nanochannel chip including: a nanochannel formed in an upper surface of the nanochannel chip and; a roof covering the nanochannel and comprising nanopores and a field enhancement structure; and a barrier disposed in the nanochannel. The method including: introducing a buffer solution including long-chain nucleic acids to the nanochannel chip; applying a voltage potential across the nanochannel chip to drive the nucleic acids through the nanochannel, towards the barrier, and to translocate the nucleic acids through nanopores adjacent to the barrier, such that bases of each of the nucleic acids pass through the field enhancement structure one base at a time and emerge onto an upper surface of the roof; detecting the Raman spectra of the bases of the nucleic acids as each base passes through the electromagnetic-field enhancement structure; and sequencing the nucleic acids based on the detected Raman spectra.

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.

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.

Methods of isolating and assaying fetal extravillous trophoblast cells, including assays of RNA of the fetal extravillous trophoblast cells according to aspects of the disclosure include obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; fixing the maternal endocervical sample in an aldehyde fixative, removing fetal extravillous trophoblast cells from the maternal endocervical sample thereby producing isolated extravillous trophoblast cells, isolating and assaying RNA from the fetal extravillous trophoblast cells.

Methods of isolating and assaying fetal extravillous trophoblast cells, including assays of RNA of the fetal extravillous trophoblast cells according to aspects of the disclosure include obtaining a maternal endocervical sample containing fetal extravillous trophoblast cells from a pregnant subject; fixing the maternal endocervical sample in an aldehyde fixative, removing fetal extravillous trophoblast cells from the maternal endocervical sample thereby producing isolated extravillous trophoblast cells, isolating and assaying RNA from the fetal extravillous trophoblast cells.