High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry
First Claim
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1. A method for sequencing DNA by detecting the identity of a dideoxynucleotide incorporated to the 3′
- end of a DNA sequencing fragment using mass spectrometry, which comprises;
(a) attaching a chemical moiety via a linker to a dideoxynucleotide to produce a labeled dideoxynucleotide;
(b) terminating a DNA sequencing reaction with the labeled dideoxynucleotide to generate a labeled DNA sequencing fragment, wherein the DNA sequencing fragment has a 3′
end and the chemical moiety is attached via the linker to the 3′
end of the DNA sequencing fragment;
(c) capturing the labeled DNA sequencing fragment on a surface coated with a compound that specifically interacts with the chemical moiety attached via the linker to the DNA sequencing fragment, thereby capturing the DNA sequencing fragment;
(d) washing the surface to remove any non-bound component;
(e) freeing the DNA sequencing fragment from the surface; and
(f) analyzing the DNA sequencing fragment using mass spectrometry so as to sequence the DNA.
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Abstract
This invention provides methods for sequencing DNA by detecting the identity of a nucleotide within a DNA sequencing fragment using mass spectrometry. The invention provides cleavable linkers for attaching a label to a dideoxynucleotide and provides labeled dideoxynucleotides. The invention also provides methods for increasing mass spectrometry resolution using linkers with different mass. The invention further provides systems for separating a labeled moiety from non-labeled components in one or more samples in solution.
160 Citations
58 Claims
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1. A method for sequencing DNA by detecting the identity of a dideoxynucleotide incorporated to the 3′
- end of a DNA sequencing fragment using mass spectrometry, which comprises;
(a) attaching a chemical moiety via a linker to a dideoxynucleotide to produce a labeled dideoxynucleotide;
(b) terminating a DNA sequencing reaction with the labeled dideoxynucleotide to generate a labeled DNA sequencing fragment, wherein the DNA sequencing fragment has a 3′
end and the chemical moiety is attached via the linker to the 3′
end of the DNA sequencing fragment;
(c) capturing the labeled DNA sequencing fragment on a surface coated with a compound that specifically interacts with the chemical moiety attached via the linker to the DNA sequencing fragment, thereby capturing the DNA sequencing fragment;
(d) washing the surface to remove any non-bound component;
(e) freeing the DNA sequencing fragment from the surface; and
(f) analyzing the DNA sequencing fragment using mass spectrometry so as to sequence the DNA. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
- end of a DNA sequencing fragment using mass spectrometry, which comprises;
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2. A method for sequencing DNA by detecting the identity of a plurality of dideoxynucleotides incorporated to the 3′
- end of different DNA sequencing fragments using mass spectrometry, which comprises;
(a) attaching a chemical moiety via a linker to a plurality of different dideoxynucleotides to produce labeled dideoxynucleotides;
(b) terminating a DNA sequencing reaction with the labeled dideoxynucleotides to generate labeled DNA sequencing fragments, wherein the DNA sequencing fragments have a 3′
end and the chemical moiety is attached via the linker to the 3′
end of the DNA sequencing fragments;
(c) capturing the labeled DNA sequencing fragments on a surface coated with a compound that specifically interacts with the chemical moiety attached via the linker to the DNA sequencing fragments, thereby capturing the DNA sequencing fragments;
(d) washing the surface to remove any non-bound component;
(e) freeing the DNA sequencing fragments from the surface; and
(f) analyzing the DNA sequencing fragments using mass spectrometry so as to sequence the DNA. - View Dependent Claims (3)
- end of different DNA sequencing fragments using mass spectrometry, which comprises;
- 25. A linker for attaching a chemical moiety to a dideoxynucleotide, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid.
- 33. A labeled dideoxynucleotide, which comprises a chemical moiety attached via a linker to a 5-position of cytosine or thymine or to a 7-position of adenine or guanine.
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42. A system for separating a chemical moiety. from other components in a sample in solution, which comprises:
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(a) a channel coated with a compound that specifically interacts with the chemical moiety, wherein the channel comprises a plurality of ends;
(b) a plurality of wells each suitable for holding the sample;
(c) a connection between each end of the channel and a well; and
(d) a means for moving the sample through the channel between wells. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55)
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- 56. A method of increasing mass spectrometry resolution between different DNA sequencing fragments, which comprises attaching different linkers to different dideoxynucleotides used to terminate a DNA sequencing reaction and generate different DNA sequencing fragments, wherein the different linkers increase mass separation between the different DNA sequencing fragments, thereby increasing mass spectrometry resolution.
Specification