Polynucleotide synthesis
2 Assignments
0 Petitions
Accused Products
Abstract
Methods of improving the kinetics of bimolecular interactions where reactants are present in low concentrations are provided. Methods of pre-amplifying one or more oligonucleotides using high concentration universal primers are provided. Methods of improving the error rate in oligonucleotide and/or polynucleotide syntheses are also provided. Methods for sequence optimization and oligonucleotides design are further provided.
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Citations
121 Claims
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1-84. -84. (canceled)
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85. An article of manufacture comprising a multiplicity of different, retrievable polynucleotides, the article comprising:
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a polynucleotide reservoir which contains a mixture of different polynucleotides comprising differing pairs of primer sequences which permit amplification of a subgroup of said different polynucleotides from said reservoir; and
plural primer reservoirs each of which contains a pair of oligonucleotide primers complementary to a pair of primer sequences of a polynucleotide in the construct reservoir.
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86. The article of claim 85 wherein the primer sequence pairs of polynucleotides in a polynucleotide reservoir are different from each other.
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87. The article of claim 85 wherein the polynucleotides comprise synthetic DNA.
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88. The article of claim 85 wherein the polynucleotides comprise genes.
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89. The article of claim 85 wherein the polynucleotides comprise multiple mutants of a wild-type sequence.
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90. The article of claim 85 wherein the polynucleotides comprise vectors.
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91. The article of claim 85 wherein at least a portion of said polynucleotides are at least one Kb long.
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92. The article of claim 85 wherein at least a portion of said polynucleotides are at least two Kb long.
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93. The article of claim 85 wherein at least a portion of said polynucleotides are at least ten Kb long.
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94. The article of claim 85 wherein at least a portion of said polynucleotides are circularized.
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95. The article of claim 85 wherein the polynucleotides comprise a polynucleotide sequence flanked by adapter sequences to facilitate manipulation of the polynucleotide sequence.
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96. The article of claim 95 wherein the adapter sequences facilitate one or more of insertion into a vector, immobilization, and identification of a function of the sequence.
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97. The article of claim 85 wherein said mixture of polynucleotides comprises one or more sequences selected from the group consisting of mammalian sequences, yeast sequences, prokaryotic sequences, plant sequences, D. melanogaster sequences, C. elegans sequences, and Xenopus sequences.
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98. The article of claim 85 wherein the mixture of different, retrievable polynucleotide constructs are independently retrievable.
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99. The article of claim 85 comprising plural polynucleotide reservoirs containing plural different polynucleotides, the polynucleotides in different reservoirs comprising an identical said pair of primer sequences.
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100-102. -102. (canceled)
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103. The article of claim 85 wherein a polynucleotide reservoir contains different polynucleotides comprising plural nested pairs of primer sequences, each of said plural nested pairs permitting amplification of a selected group of polynucleotides in said reservoir or of individual ones of said different polynucleotides therein.
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104. The article of claim 85 comprising 102 different polynucleotides.
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105. The article of claim 85 comprising 103 different polynucleotides.
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106. The article of claim 85 comprising 104 different polynucleotides.
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107. The article of claim 85 comprising 105 different polynucleotides.
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108. The article of claim 85 comprising 106 different polynucleotides.
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109. An article of manufacture comprising a package containing a multiplicity of different, retrievable polynucleotides, the article comprising:
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a polynucleotide reservoir which contains a mixture of different polynucleotides at least some of which comprise plural nested pairs of primer sequences, each of said plural nested pairs permitting amplification of a selected group of polynucleotides in said reservoir or of individual ones of said different polynucleotides therein; and
plural primer reservoirs each of which contains a pair of oligonucleotide primers complementary to a pair of primer sequences of a polynucleotide in said construct reservoir.
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110-119. -119. (canceled)
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120. A method of obtaining a polynucleotide of choice comprising the steps of:
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providing plural construct reservoirs containing mixtures of identified synthesized polynucleotides comprising plural nested pairs of primer sequences which permit amplification of selected ones of said polynucleotides from a said reservoir, the combination of primer pairs of a polynucleotide in a said reservoir being different from other pairs of primer sequence of other polynucleotides in said reservoir;
providing plural primer reservoirs each of which contains a pair of oligonucleotide primers complementary to a pair of primer sequences of a polynucleotide in a said construct reservoirs;
conducting a first amplification procedure in a first amplification mixture comprising an aliquot of a said mixture of polynucleotides retrieved from a selected said construct reservoir and a pair of primers complementary to an outer nested pair of primer sequences retrieved from one or more primer reservoirs; and
conducting a second amplification procedure in a second amplification mixture comprising an aliquot of amplicons retrieved from said first amplification mixture and a pair of primers complementary to an inner nested pair of primer sequences retrieved from one or more primer reservoirs.
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121-124. -124. (canceled)
Specification