Direct nucleic acid sequencing method
OwnerShenglong Zhang, Ning Zhanginventors
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Abstract
The present disclosure relates generally to novel methods for nucleic acid sequencing. Specifically, the invention relates to a liquid chromatography-mass-spectrometry (LC-MS) based technique for direct sequencing of RNA without cDNA. The technique allows one to simultaneously read an RNA sequence with single nucleotide resolution while determining the presence, type and location of a wide spectrum of RNA modifications.
Claims35
An RNA sequencing method, for determining the primary RNA sequence and the presence/identification/location of RNA modifications, comprising the steps of:
- (i) labeling of the 5′ and/or 3′ end of the RNA;
- (ii) random degradation of the RNA;
- (iii) optionally, physical separation of resultant RNA fragments based on 5′ and 3′ end labeling;
- (iv) separation and detection of the resultant RNA fragment properties;
- and (v) data analysis resulting in sequence/modification identification.
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7-10. (canceled)
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An RNA sequencing method comprising the steps of:
- (i) labeling of the 5′ and/or 3′ end of the RNA with a moiety that increases the hydrophobicity of the RNA fragments thereby increasing the retention time of degraded RNA fragments;
- (ii) random degradation of the RNA;
- (iii) separation and detection of the resultant RNA fragment properties;
- and (iv) data analysis resulting in sequence/modification identification.
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A DNA sequencing method comprising the steps of:
- (i) affinity labeling of the 5′ and/or 3′ end of the DNA;
- (ii) random degradation of the DNA into mass ladders;
- (iii) optionally, physical separation of resultant DNA fragments based on an affinity interaction;
- (iv) measurement of resultant DNA fragments using reverse-phase high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) or other separation methods coupled with mass spectrometry;
- and (v) MS data analysis resulting in sequence/modification identification.
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An RNA sequencing method wherein the RNA is ψ-containing, comprising the steps of:
- (i) treatment of RNA to be sequenced with CMC;
- (ii) affinity labeling of the 5′ and 3′ end of the RNA;
- (iii) random degradation of the RNA;
- (iv) optionally, physical separation of resultant RNA fragments based on an affinity interaction;
- (v) measurement of resultant RNA fragments using reverse-phase high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) or other separation methods coupled with mass spectrometry;
- and (v) MS data analysis resulting in sequence/modification identification.
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39-40. (canceled)
Dependencies are inferred from the claim text.