Sample preparation for LC-MS based sequence mapping of nucleic acids
AssigneeWaters Technologies Corporation
InventorsMatthew A Lauber, Xiaoxiao Liu
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Abstract
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.
Claims14
A method for sequence analysis of a target nucleic acid present in a sample comprising the steps of:
- a. treating the sample with a nuclease free buffer comprising a first optional non-ionic or anionic denaturant and an optional metal-chelation agent to denature the target nucleic acid;
- b. incubating the sample with a nuclease enzyme for a predetermined amount of time, wherein the nuclease enzyme is capable of fragmenting the target nucleic acid to produce a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of the target nucleic acid;
- c. inactivating the fragmenting capability of the nuclease enzyme by addition of a second non-ionic or anionic denaturant and an optional disulfide bond reducing agent;
- d. subjecting the set of nucleic acid fragments to a single-mode or a multiple mode of chromatography followed by a mass spectrometry (MS) method;
- and
- e. generating mass signals of the set of nucleic acid fragments by the mass spectrometry method whereby determining the sequence of the target nucleic acid from the mass signals.
Dependencies are inferred from the claim text.