Methods and compositions for determining the sequence of nucleic acid molecules
First Claim
1. A method comprising:
- (a) providing nucleic acid fragments, each fragment having cleavably attached thereto a mass tag;
(b) separating the tagged fragments on the basis of fragment charge, sequential length, size or shape;
(c) placing a charge on the tag and cleaving the tag from the fragment, where the charge is placed on the tag prior to, during, or after cleaving the tag from the fragment; and
(d) characterizing each tag by mass spectrometry, wherein at least one tagged nucleic acid fragment is a compound of the formula;
Tms-L-Xwherein,Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a Tms-containing moiety to be cleaved from the remainder of the compound, wherein the Tms-containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and
X is a nucleic acid fragment attached to L at other than the 3′
end of the nucleic acid fragment;
with the provisos that the compound is not bonded to a solid support nor has a mass of less than 250 daltons.
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Abstract
Methods and compounds, including compositions therefrom, are provided for determining the sequence of nucleic acid molecules. The methods permit the determination of multiple nucleic acid sequences simultaneously. The compounds are used as tags to generate tagged nucleic acid fragments which are complementary to a selected target nucleic acid molecule. Each tag is correlative with a particular nucleotide and, in a preferred embodiment, is detectable by mass spectrometry. Following separation of the tagged fragments by sequential length, the tags are cleaved from the tagged fragments. In a preferred embodiment, the tags are detected by mass spectrometry and the sequence of the nucleic acid molecule is determined therefrom. The individual steps of the methods can be used in automated format, e.g., by the incorporation into systems.
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Citations
30 Claims
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1. A method comprising:
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(a) providing nucleic acid fragments, each fragment having cleavably attached thereto a mass tag; (b) separating the tagged fragments on the basis of fragment charge, sequential length, size or shape; (c) placing a charge on the tag and cleaving the tag from the fragment, where the charge is placed on the tag prior to, during, or after cleaving the tag from the fragment; and (d) characterizing each tag by mass spectrometry, wherein at least one tagged nucleic acid fragment is a compound of the formula;
Tms-L-Xwherein, Tms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic group which allows a Tms-containing moiety to be cleaved from the remainder of the compound, wherein the Tms-containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and X is a nucleic acid fragment attached to L at other than the 3′
end of the nucleic acid fragment;with the provisos that the compound is not bonded to a solid support nor has a mass of less than 250 daltons. - 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, 26, 27, 28, 29, 30)
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Specification