Solid phase sequencing of double-stranded nucleic acids
First Claim
1. A method for sequencing a target nucleic acid comprising the steps of:
- (a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;
(b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, wherein each probe contains a variable sequence within the single-stranded region, and for each complex;
(i) ligating a single strand of the fragment to an adjacent single strand of the probe; and
(ii) extending the unligated strand of the complex by strand displacement polymerization using the ligated strand as a template; and
(c) determining the molecular weights of the extended strands; and
(d) determining the sequence of the target from the molecular weights of the extended strands, wherein the extended strand contains mass-modifying functionalities.
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Accused Products
Abstract
Methods for detecting and sequencing of target double-stranded nucleic acid molecules, nucleic acid probes and arrays of probes useful in these methods, and kits and systems that contain these probes aer provided. The 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 include 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.
134 Citations
28 Claims
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1. A method for sequencing a target nucleic acid comprising the steps of:
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(a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;
(b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, wherein each probe contains a variable sequence within the single-stranded region, and for each complex;
(i) ligating a single strand of the fragment to an adjacent single strand of the probe; and
(ii) extending the unligated strand of the complex by strand displacement polymerization using the ligated strand as a template; and
(c) determining the molecular weights of the extended strands; and
(d) determining the sequence of the target from the molecular weights of the extended strands, wherein the extended strand contains mass-modifying functionalities. - 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)
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Specification