Methods and compositions for determining the sequence of nucleic acid molecules
First Claim
1. A method for determining the sequence of a nucleic acid molecule, comprising:
- (a) generating tagged nucleic acid fragments which are complementary to a selected target nucleic acid molecule, wherein a tag is an organic moiety that is correlative with a particular nucleotide and detectable by non-fluorescent spectrometry or potentiometry;
(b) separating the tagged fragments by sequential length;
(c) cleaving the tags from the tagged fragments; and
(d) detecting the tags by non-fluorescent spectrometry or potentiometry, and therefrom determining the sequence of the nucleic acid molecule.
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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.
392 Citations
58 Claims
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1. A method for determining the sequence of a nucleic acid molecule, comprising:
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(a) generating tagged nucleic acid fragments which are complementary to a selected target nucleic acid molecule, wherein a tag is an organic moiety that is correlative with a particular nucleotide and detectable by non-fluorescent spectrometry or potentiometry;
(b) separating the tagged fragments by sequential length;
(c) cleaving the tags from the tagged fragments; and
(d) detecting the tags by non-fluorescent spectrometry or potentiometry, and therefrom determining the sequence of the nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 28, 29, 30, 31, 32)
wherein G is (CH2)1-6 wherein a hydrogen on one and only one of the CH2 groups of each G is replaced with —
(CH2)c-Amide-T4;
T2 and T4 are organic moieties of the formula C1-25N0-9S0-3P0-3Hα
Fβ
Iδ
wherein the sum of α
, β and
δ
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, S and P atoms;
Amide is
R1 is hydrogen or C1-10 alkyl;
c is an integer ranging from 0 to 4;
X is defined according to claim 1; and
n is an integer ranging from 1 to 50 such that when n is greater than 1, G, c, Amide, R1 and T4 are independently selected.
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29. A compound according to claim 28 having the formula:
-
wherein T5 is an organic moiety of the formula C1-25N0-9O0-9S0-3P0-3Hα
Fβ
Iδ
wherein the sum of α
, β and
δ
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, S and P atoms; and
T5 includes a tertiary or quaternary amine or an organic acid; and
m is an integer ranging from 0-49.
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-
30. A compound according to claim 28 having the formula:
-
wherein T5 is an organic moiety of the formula C1-25N0-9O0-9S0-3P0-3Hα
Fβ
Iδ
wherein the sum of α
, β and
δ
is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, O, S and P atoms; and
T5 includes a tertiary or quaternary amine or an organic acid; and
m is an integer ranging from 0-49.
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31. A compound according to any one of claims 29 and 30 wherein -Amide-T5 is selected from:
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32. A compound according to any of claims 29 and 30 wherein -Amide-T5 is selected from:
- 15. A compound of the formula:
- 34. A composition comprising a plurality of compounds of the formula:
- 44. A composition comprising a plurality of sets of compounds, each set of compounds having the formula:
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48. A kit for DNA sequencing analysis comprising a plurality of container sets, each container set comprising at least five containers, wherein a first container contains a vector, a second, third, fourth and fifth containers contain compounds of the formula:
-
Tms—
L—
MOIwherein, 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
MOI is a nucleic acid fragment wherein L is conjugated to the MOI at a location other than the 3′
end of the MOI;
such thatthe MOI for the second, third, fourth and fifth containers is identical and complementary to a portion of the vector within the set of containers, and the Tms group within each container is different from the other Tms groups in the kit. - View Dependent Claims (49, 50)
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- 51. An apparatus for determining the sequence of a nucleic acid molecule in a sample, the sample including tagged nucleic acid fragments having nucleic acid fragments and tags attached to the nucleic acid fragments, comprising a separation apparatus that separates tagged nucleic acid fragments, a cleavage apparatus that receives separated tagged cleaves nucleic acid fragments and the tags from the nucleic acid fragments, each tag being correlative with a particular nucleotide of the nucleic acid fragment and detectable by electrochemical detection, and an apparatus for electrochemical detection that receives and detects electrochemical signatures of the tags.
- 56. An apparatus for determining the sequence of a nucleic acid molecule in a sample, the sample including tagged nucleic acid fragments having nucleic acid fragments and tags attached to the nucleic acid fragments, comprising a separation apparatus that separates tagged nucleic acid fragments, a cleavage apparatus that receives separated tagged nucleic acid fragments and cleaves from the nucleic acid fragments, each tag being correlative with a particular nucleotide of the nucleic acid fragment and detectable by mass spectrometry, a mass spectrometer that receives the tags and detects a mass of a tag, and a data processor that correlates the mass of a tag to a particular nucleotide and to a specific sample.
Specification