Sequencing duplex DNA by mass spectroscopy
First Claim
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1. A method of sequencing a nucleic acid of interest comprising:
- providing four populations of pluralities of duplex nucleic acids, each nucleic acid having a common end and a terminal base at the other end, and a length corresponding to the position of the terminal base in the nucleic acid of interest, the duplex nucleic acids having an ionization target, and a detection label associated with the termination base, ionizing the ionizing targets of the populations of duplex nucleic acid with an ionizing agent, fractionating the populations of duplex nucleic acid using mass spectroscopy, for each duplex nucleic acid, resolving a single ionization state, identifying the terminal base by means of the detection label, and determining the sequence length based on mass.
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Abstract
For the determination of masses of macromolecular analytes with particular application to DNA sequencing by mass spectroscopy, novel strategies of sample preparation and labeling decrease macromolecule breakage, improve identification of population members, aid attainment of a single charge state for the heterogeneous analyte inputs, and increase the sensitivity of detection of the fractionated macromolecules.
77 Citations
40 Claims
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1. A method of sequencing a nucleic acid of interest comprising:
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providing four populations of pluralities of duplex nucleic acids, each nucleic acid having a common end and a terminal base at the other end, and a length corresponding to the position of the terminal base in the nucleic acid of interest, the duplex nucleic acids having an ionization target, and a detection label associated with the termination base, ionizing the ionizing targets of the populations of duplex nucleic acid with an ionizing agent, fractionating the populations of duplex nucleic acid using mass spectroscopy, for each duplex nucleic acid, resolving a single ionization state, identifying the terminal base by means of the detection label, and determining the sequence length based on mass. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40)
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26. A method of determining the mass of a macromolecule comprising:
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providing the macromolecule with an ionization target and a detection label, ionizing the ionizing targets with an ionizing agent to provide essentially a single ionization state, subjecting the macromolecule to fractionation by mass spectroscopy, detecting the detection label and determining the mass of the macromolecule.
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38. A device for sequencing DNA comprising:
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means for producing four populations of pluralities of duplex nucleic acids, each nucleic acid having a common end and a terminal base at the other end, and a length corresponding to the position of the terminal base in the nucleic acid of interest, the duplex nucleic acids having an ionization target, and a detection label associated with the termination base, means for ionizing the ionizing targets of the populations of duplex nucleic acid with an ionizing agent, means for fractionating the populations of duplex nucleic acid using mass spectroscopy, means for detecting the detection label on the terminal base of each duplex nucleic acid, and means for determining the sequence length based on mass.
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39. A population of duplex DNA molecules of lengths greater than about 50 bases, corresponding to the sequence of a nucleic acid of interest, each molecule having a common end and a terminal base at the other end, and a length corresponding to the position of the terminal base in the nucleic acid of interest, and each molecule having an ionization target and a detection label associated with the terminal base, each molecule being susceptible to ionization to produce essentially a single charge state for that length.
Specification