Methods of synthesizing labeled polynucleotides by ligation of multiple oligomers
First Claim
1. A method comprising:
- a) providing a reaction mixture comprising a single stranded nucleic acid template, a primer having at least 15 bases which is complementary to a portion of the single stranded nucleic acid template and a plurality of oligonucleotide 5'"'"'-monophosphates wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases and wherein each of the oligonucleotide 5'"'"'-monophosphates is labeled;
b) hybridizing the primer with the template under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to form a primer-template hybrid having a single stranded region and a double stranded region;
c) ligating more than one of the plurality of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the primer in one continuous process under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to extend the double stranded region and synthesize a labeled complementary nucleic acid strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer.
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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.
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Citations
23 Claims
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1. A method comprising:
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a) providing a reaction mixture comprising a single stranded nucleic acid template, a primer having at least 15 bases which is complementary to a portion of the single stranded nucleic acid template and a plurality of oligonucleotide 5'"'"'-monophosphates wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases and wherein each of the oligonucleotide 5'"'"'-monophosphates is labeled; b) hybridizing the primer with the template under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to form a primer-template hybrid having a single stranded region and a double stranded region; c) ligating more than one of the plurality of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the primer in one continuous process under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to extend the double stranded region and synthesize a labeled complementary nucleic acid strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for synthesizing a labeled double stranded nucleic acid wherein both strands are labeled comprising:
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a) providing a reaction mixture comprising a double stranded nucleic acid template, a first primer which is complementary to a region of a first strand of the template, a second primer which is complementary to a region of a second strand of the template, wherein both primers have at least 15 bases, and a plurality of labeled oligonucleotide 5'"'"'-monophosphates wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases and wherein each oligonucleotide 5'"'"'-monophosphate is labeled; b) separating the first and second strands of the double stranded nucleic acid; c) hybridizing the first and second primers with the separated strands under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to form first and second primer-template hybrids; d) ligating more than one of the plurality of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto each primer-template hybrid in one continuous process under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to extend the first and second primers thereby producing double stranded nucleic acid, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer; and
e) repeating steps b-d at least once, thereby synthesizing a labeled double stranded nucleic acid wherein both strands are labeled.
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Specification