Methods of synthesizing and amplifying 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'"'"'monophoshate consists of not more than 10 bases;
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; and
c) ligating more than one of the plurality of 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'"'"'-monophospates, to extend the double stranded region and synthesize a 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
59 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'"'"'monophoshate consists of not more than 10 bases; 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; and c) ligating more than one of the plurality of 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'"'"'-monophospates, to extend the double stranded region and synthesize a 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, 24, 25, 26, 27, 29)
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18. A method for synthesizing an immobilized single stranded nucleic acid on a solid support, the nucleic acid having a region with a base sequence which is complementary to a portion of the base sequence of a test single stranded nucleic acid comprising;
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a) providing a reaction mixture comprising the test single stranded nucleic acid, a capture probe/primer having at least 15 bases which is complementary to a portion of the test single stranded nucleic acid and which is immobilized on the solid support, and a plurality of oligonucleotide 5'"'"'-monophosphates, wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases; b) hybridizing the capture probe/primer with the test single stranded nucleic acid under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates, to capture the test single stranded nucleic acid and form a captured probe-test nucleic acid hybrid having a single stranded region and a double stranded region; c) ligating more than one of the plurality of oligonucleotide 5'"'"'-monophosphates to the capture probe/primer under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates, to extend the double stranded region wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer; d) removing the oligonucleotide 5'"'"'-monophosphates which are not ligated; and e) denaturing the captured probe-test nucleic acid hybrid having an extended double stranded region to remove the test nucleic acid and produce the immobilized single stranded nucleic acid.
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19. A method for amplifying the amount of a portion of a double stranded nucleic acid having a first strand and a second strand comprising;
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a) providing in a reaction mixture the double stranded nucleic acid, a first primer having at least 15 bases which is complementary to a region of the first strand, a second primer having at least 15 bases which is complementary to a region of the second strand wherein the first and second regions define the ends the portion of the double stranded nucleic acid to be amplified, and a plurality of oligonucleotide 5'"'"'-monophosphates, wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases; 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 primers but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates; d) ligating onto each of the hybridized first and second primers more than one of the plurality of oligonucleotide 5'"'"'-monophosphates in a contiguous manner in one continuous process under conditions which permit stable hybridization of the primers but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates, to extend the first and second primers, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primers, thereby producing double stranded nucleic acid; and e) repeating steps b-d to increase the amount of the portion of the double stranded nucleic acid. - View Dependent Claims (20, 21, 22, 23, 28, 30)
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31. A method comprising:
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a) providing a reaction mixture comprising a single stranded nucleic acid template having a segment of known sequence, a primer having at least 15 bases which is complementary to a portion of the segment of known sequence, and a set of oligonucleotide 5'"'"'-monophosphates, wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases, and wherein the oligonucleotide 5'"'"'-monophosphates are selected to be complementary to a part of the segment of known sequence of the template adjacent to the portion of the segment of known sequence to which the primer is complementary; 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 the set of 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 complementary nucleic acid strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 59)
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51. A method for synthesizing an immobilized single stranded nucleic acid on a solid support comprising:
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a) providing a reaction mixture comprising a test single stranded nucleic acid having a segment of known sequence, a capture probe/primer having at least 15 bases which is complementary to a portion of the segment of known sequence and which is immobilized on the solid support, and a set of oligonucleotide 5'"'"'-monophosphates, wherein each oligonucleotide 5'"'"'-monophoshate consists of not more than 10 bases, and wherein the oligonucleotide 5'"'"'-monophospates are selected to be complementary to a part of the segment of known sequence of the test single stranded nucleic acid adjacent to the portion of the segment of known sequence to which the primer is complementary; b) hybridizing the capture probe/primer with the test single stranded nucleic acid under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to form a captured probe-test nucleic acid hybrid; c) ligating the set of oligonucleotide 5'"'"'-monophosphates onto the capture probe/primer under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to extend the capture probe/primer wherein ligation of oligonucleotide 5'"'"'-monophosphates; d) removing the oligonucleotide 5-monophosphates which are not ligated; and e) denaturing the captured probe-test nucleic acid hybrid to remove the test nucleic acid and produce the immobilized single stranded nucleic acid.
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52. A method for amplifying the amount of a segment of a double stranded nucleic acid of known sequence, the segment having a first strand and a second strand comprising:
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a) providing a reaction mixture comprising the double stranded nucleic acid, a first primer having at least 15 bases which is complementary to a first portion of the first strand, a second primer having at least 15 bases which is complementary to a first portion of the second strand, wherein the first portion of the first strand and the first portion of the second strand define the ends of the segment of the double stranded nucleic acid to be amplified, a first set of oligonucleotide 5'"'"'-monophosphates selected to be complementary to a second portion of the first strand, wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases, and a second set of oligonucleotide 5'"'"'-monophosphates selected to be complementary to a second portion of the second strand, wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases; 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; d) ligating the first set of oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the hybridized first primer and ligating the second set of oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the hybridized second primer in one continuous process to extend the first and second primers thereby producing the segment of the double stranded nucleic acid, wherein ligation of oligonucleotide 5'"'"'-phosphates is performed under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates and only wherein ligation occurs in the presence of the hybridized primers; and e) repeating steps b-d to increase the amount of the segment of the double stranded nucleic acid of known sequence. - View Dependent Claims (53, 54, 55, 56, 57, 58)
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Specification