Solid phase sequencing of biopolymers
First Claim
1. A method for sequencing a target nucleic acid molecule, comprising:
- fragmenting the target nucleic acid molecule to produce a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of the target nucleic acid;
hybridizing the set to an array of nucleic acid probes to form a target array of nucleic acid molecules, wherein;
each probe comprises a single-stranded portion comprising a variable region;
andthe array comprises a collection of the probes with sufficient sequence diversity in the variable regions to hybridize all of the target sequence with complete or nearly complete discrimination;
releasing the nucleic acid fragments hybridized to the probes, thereby producing released fragments;
generating mass signals of the released fragments by mass spectrometry;
wherein the released fragments are not labeled; and
determining the sequence of the target nucleic acid from the mass signals.
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Abstract
This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.
177 Citations
77 Claims
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1. A method for sequencing a target nucleic acid molecule, comprising:
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fragmenting the target nucleic acid molecule to produce a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of the target nucleic acid; hybridizing the set to an array of nucleic acid probes to form a target array of nucleic acid molecules, wherein; each probe comprises a single-stranded portion comprising a variable region; and the array comprises a collection of the probes with sufficient sequence diversity in the variable regions to hybridize all of the target sequence with complete or nearly complete discrimination; releasing the nucleic acid fragments hybridized to the probes, thereby producing released fragments; generating mass signals of the released fragments by mass spectrometry; wherein the released fragments are not labeled; and determining the sequence of the target nucleic acid from the mass signals. - 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77)
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Specification