METHOD FOR CONFIRMING THE SEQUENCE OF A NUCLEAN ACID

Abstract

A method for confirming the sequence of a nucleic acid is provided, comprising: (i) hydrolytic cleavage of predetermined phosphodiester bonds in the nucleic acid to generate a plurality of oligonucleotides; (ii) ionization of the plurality of oligonucleotides to provide a gas phase of ionized oligonucleotides, wherein a majority of the ionized oligonucleotides are singly charged; (iii) isolation of the ionized oligonucleotides using a plurality of mass-to-charge ratio (m / z) windows; (iv) detection of the isolated ionized oligonucleotides of each m / z window using a high-resolution mass spectrometer (HRMS) with a resolution of more than 30.000 at m / z 200, thereby obtaining a mass spectrum; (v) Confirming the sequence of the nucleic acid by comparing the mass spectrum obtained in step (iv) with a mass spectrum determined in silico after the same hydrolytic cleavage of the nucleic acid as in step (i), the confirmation comprising a comparison of the signal intensities of one or more mass envelopes resulting from the ionized oligonucleotides.

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