Methods of sequencing polynucleotides by ligation of multiple oligomers
First Claim
1. A method for determining the base sequence of a portion of a single stranded nucleic acid by determining the base sequence of a complementary strand comprising the steps of:
- a) providing a reaction mixture comprising a single stranded nucleic acid, a capture probe/primer which is complementary to a portion of the single stranded nucleic acid, and a plurality of labeled identical length oligonucleotide 5'"'"'-monophsphates wherein each opligonucleotide 5'"'"'-monophophate compxises a label that is specific for and identifies that oligonucleotide 5'"'"'-monophosphate;
b) hybridizing the capture probe/primer with the single stranded nucleic acid to form a captured probe-nucleic acid hybrid;
c) ligating more than one of the plurality of the oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the capture probe/primer in one continuous process to synthesize the complementary strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer;
d) removing all nonligated oligonucleotide 5'"'"'-monophosphates;
e) detecting the plurality of labels which were attached in the ligation;
f) relating the plurality of detected labels to the identity of their corresponding oligonucleotide 5'"'"'-monophosphates; and
g) determining the base sequence of the portion of the nucleic acid from the identity of the plurality of oligonucleotide 5'"'"'-monophosphates identified in step f.
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Accused Products
Abstract
Methods of synthesizing polynucleotides are disclosed involving the simultaneous ligation of a set of oligomer 5'"'"'-phosphates onto a template-bound primer. The set of these oligomers can be preselected to contain oligomers which are complementary to the template strand or the oligomers can be supplied as a library and allowed to self select. The synthesis by ligation can proceed unidirectionally or bidirectionally from the primer and can be used to synthesize both strands simultaneously by the use of two primers. The ligation is preferably performed with a ligase enzyme. The methods of synthesis are useful in a variety of applications, including cloning, amplification, labeling, diagnostic assays, mutation analysis and screening, gene expression monitoring and sequence analysis.
115 Citations
43 Claims
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1. A method for determining the base sequence of a portion of a single stranded nucleic acid by determining the base sequence of a complementary strand comprising the steps of:
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a) providing a reaction mixture comprising a single stranded nucleic acid, a capture probe/primer which is complementary to a portion of the single stranded nucleic acid, and a plurality of labeled identical length oligonucleotide 5'"'"'-monophsphates wherein each opligonucleotide 5'"'"'-monophophate compxises a label that is specific for and identifies that oligonucleotide 5'"'"'-monophosphate; b) hybridizing the capture probe/primer with the single stranded nucleic acid to form a captured probe-nucleic acid hybrid; c) ligating more than one of the plurality of the oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the capture probe/primer in one continuous process to synthesize the complementary strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer; d) removing all nonligated oligonucleotide 5'"'"'-monophosphates; e) detecting the plurality of labels which were attached in the ligation; f) relating the plurality of detected labels to the identity of their corresponding oligonucleotide 5'"'"'-monophosphates; and g) determining the base sequence of the portion of the nucleic acid from the identity of the plurality of oligonucleotide 5'"'"'-monophosphates identified in step f. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for determining the base sequence of a portion of a single stranded nucleic acid by determining the base sequence of a complementary strand comprising the steps of:
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a) providing a capture probe/primer which is complementary to a portion of the single stranded nucleic acid; b) forming a plurality of sets of labeled identical length oligonucleotide 5'"'"'-monophosphates, each oligonucleotide 5'"'"'-monophosphate comprising a label that is specific for and identifies that oligonucleotide 5'"'"'-monophosphhate and each set containing a plurality of oligonucleotide 5'"'"'-monophosphates wherein one labeled oligonucleotide 5'"'"'-monophosphate which is present in all other sets is excluded from each set; c) for each set of labeled oligonucleotide 5'"'"'-monophosphates, hybridizing the capture probe/primer with the single stranded nucleic acid; d) for each set of labeled oligonucleotide 5'"'"'-monophosphates, contacting the set with the captured hybrid in a separate reaction; e) ligating each set of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the capture probe/primer in one continuous process, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer; f) removing all nonligated oligonucleotide 5'"'"'-mononophosphates in each set; g) for each set, identifying all labels which were attached in the ligation, thereby determining the number and base sequence of all oligonucleotide 5'"'"'-monophosphates ligated in each set and determining the relative position of the excluded oligonucleotide 5'"'"'-monophosphate in the base sequence of the complementary strand; and h) deducing the base sequence of the portion of the single stranded nucleic acid from either the relative positions of each of the excluded oligonucleotide 5'"'"'-monophosphates in the comlementary strand, or the number and base sequence of ligated oligonucleotide 5'"'"'-monophosphates of all sets combined. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for determining the sequence of a portion of a single stranded nucleic acid by determining the base sequence of a complementary strand comprising the steps of:
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a) providing a capture probe/primer which is complementary to a portion of the single stranded nucleic acid; b) forming a plurality of sets of labeled identical length oligonucleotide 5'"'"'-monophosphates, each oligonucleotide 5'"'"'-monophosphate having the same detectable label and each set containing a plurality of oligonucleotide 5'"'"'-monophosphates wherein one labeled oligonucleotide 5'"'"'-monophosphate which is present in all other sets is excluded from each set; c) hybridizing the capture probe/primer with the single stranded nucleic acid to form a captured probe-nucleic acid hybrid; d) for each set of labeled oligonucleotide 5'"'"'-monophosphates, contacting the set with the captured hybrid in a separate reaction; e) ligating each set of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the capture probe/primer in one continuous process, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer; f) removing all nonligated oligonucleotide 5'"'"'-monophosphates in each set; g) determining the number of labels which were attached in the ligation in each set thereby determining the relative position of the excluded oligonucleotide 5'"'"'-monophosphate in the base sequence of the complementary strand; and h) deducing the base sequence of the portion of the single stranded nubleic acid from the relative positions of each of the excluded oligonucleotide 5'"'"'-monophosphates in the complementary strand. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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Specification