NUCLEIC ACID SEQUENCE DETECTION BY ENDONUCLEASE DIGESTION AND MASS SPECTROMETRY
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.
1 . A method of genetic analysis, comprising:
(a) providing a complex mixture comprising a plurality of end-blocked polynucleotides;
(b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that cleaves a target polynucleotide comprising a sequence of interest to produce, if the sequence of interest is present in the complex mixture, a polynucleotide that comprises:
the sequence of interest and
at least one ligatable end generated by endonuclease cleavage;
(c) ligating a moiety to a ligatable end of the polynucleotide under conditions in which the moiety cannot be ligated to a polynucleotide that lacks a ligatable end to produce a moiety-target polynucleotide construct;
(d) detecting, using mass spectrometry, the moiety-target polynucleotide construct or a transcription or translation product produced from the moiety-target polynucleotide;
(e) correlating the detection of the moiety-target polynucleotide with the presence of the sequence of interest in the complex mixture.
2 . A method of genetic analysis, comprising:
(a) providing a complex mixture comprising a plurality of end-blocked polynucleotides;
(b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that cleaves a target polynucleotide comprising a sequence of interest to produce, if the sequence of interest is present in the complex mixture, a polynucleotide that comprises:
the sequence of interest and
at least one ligatable end generated by endonuclease cleavage;
(c) ligating a moiety to a ligatable end of the polynucleotide under conditions in which the moiety cannot be ligated to a polynucleotide that lacks a ligatable end to produce a moiety-target polynucleotide construct, wherein the moiety is a nucleotide adaptor comprising an RNA promoter and the moiety-target polynucleotide construct is configured so that transcription initiated at the promoter transcribes at least a portion of the sequences of interest to produce RNA products;
(d) translating the RNA products to produce a polypeptide or mixture of polypeptides;
(e) detecting, using mass spectrometry, the polypeptide or mixture of polypeptides; and
(f) correlating the detection of the polypeptide or mixture of polypeptides with the presence of the sequence of interest in the complex mixture.
3 . The method of claim 2 wherein the in vitro transcription and translation are coupled.
4 . A method of genetic analysis, comprising:
(a) providing a complex mixture comprising a plurality of end-blocked polynucleotides;
(b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that cleaves a target polynucleotide comprising a sequence of interest to produce, if the sequence of interest is present in the complex mixture, a polynucleotide that comprises:
the sequence of interest and
at least one ligatable end generated by endonuclease cleavage;
(c) ligating a moiety to a ligatable end of the polynucleotide under conditions in which the moiety cannot be ligated to a polynucleotide that lacks a ligatable end to produce a moiety-target polynucleotide construct, wherein the moiety is a nucleotide adaptor comprising an RNA promoter and the moiety-target polynucleotide construct is configured so that transcription initiated at the promoter transcribes at least a portion of the sequences of interest to produce RNA products;
(d) detecting, using mass spectrometry, the RNA products; and
(e) correlating the detection of the RNA products with the presence of the sequence of interest in the complex mixture.
5 . A method of genetic analysis, comprising:
(a) providing a complex mixture comprising a plurality of end-blocked polynucleotides;
(b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that cleaves a target polynucleotide comprising a sequence of interest to produce, if the sequence of interest is present in the complex mixture, a polynucleotide that comprises:
the sequence of interest and
at least one ligatable end generated by endonuclease cleavage;
(c) ligating a moiety to a ligatable end of the polynucleotide under conditions in which the moiety cannot be ligated to a polynucleotide that lacks a ligatable end to produce a moiety-target polynucleotide construct, wherein the moiety is a an adaptor linked to a mass label to produce adaptored polynucleotides linked to the mass label;
(d) detecting, using mass spectrometry, the RNA adaptored polynucleotides linked to the mass label; and
(e) correlating the detection of the adaptored polynucleotides linked to the mass label with the presence of the sequence of interest in the complex mixture.
6 . The method of claim 1 in which the nucleic acid-directed endonuclease is a CRISPR-associated protein (Cas) and a guide RNA.
7 . The method of claim 1 in which the sequence of interest is a gene sequence of a strain of bacteria.