Abrégé

The invention provides methods for synthesizing a product DNA molecule of any DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence.

Classes IPC  ?

• C12P 19/34 - Polynucléotides, p. ex. acides nucléiques, oligoribonucléotides
• C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
• C12Q 1/6862 - Réaction en chaine par ligase [LCR]

Abrégé

The invention provides methods for synthesizing a product DNA molecule of any possible DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence. In any embodiment the product DNA molecule has an error rate of less than 1 error per 2,000 nucleotides.

Classes IPC  ?

• C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
• C12Q 1/6844 - Réactions d’amplification d’acides nucléiques
• C12Q 1/6862 - Réaction en chaine par ligase [LCR]
• C12N 9/22 - Ribonucléases
• C12P 19/34 - Polynucléotides, p. ex. acides nucléiques, oligoribonucléotides

Abrégé

The invention provides methods of synthesizing a product DNA molecule having a desired and/or defined sequence. The methods involve annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide. After annealing, at least one long oligonucleotide bound to an anchor strand abuts at least one short oligonucleotide bound to the same anchor strand. The anchor strand has one or more non-standard nucleotides, and optionally one or more degenerate nucleotides. The method involves ligating the abutting at least one long oligonucleotide and at least one short oligonucleotide to form a dsDNA molecule. The invention also provides methods of synthesizing DNA molecules by assembling oligonucleotide members of a library that contains less than 20,000 members that can be assembled into all possible DNA sequences.

Classes IPC  ?

• C12N 15/11 - Fragments d'ADN ou d'ARNLeurs formes modifiées
• C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
• C40B 40/06 - Bibliothèques comprenant des nucléotides ou des polynucléotides ou leurs dérivés

Abrégé

The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.

Classes IPC  ?

• C12N 15/09 - Technologie d'ADN recombinant
• C12N 15/11 - Fragments d'ADN ou d'ARNLeurs formes modifiées
• C12P 19/34 - Polynucléotides, p. ex. acides nucléiques, oligoribonucléotides
• C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques

Abrégé

The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.

Classes IPC  ?

• C07K 14/28 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Vibrionaceae (F)
• C12N 1/21 - BactériesLeurs milieux de culture modifiés par l'introduction de matériel génétique étranger
• C12N 15/09 - Technologie d'ADN recombinant
• C12N 15/31 - Gènes codant pour des protéines microbiennes, p. ex. entérotoxines
• C12N 15/63 - Introduction de matériel génétique étranger utilisant des vecteursVecteurs Utilisation d'hôtes pour ceux-ciRégulation de l'expression

Abrégé

The invention provides methods of synthesizing a product DNA molecule having a desired and/or defined sequence. The methods involve annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide. After annealing, at least one long oligonucleotide bound to an anchor strand abuts at least one short oligonucleotide bound to the same anchor strand. The anchor strand has one or more non-standard nucleotides, and optionally one or more degenerate nucleotides. The method involves ligating the abutting at least one long oligonucleotide and at least one short oligonucleotide to form a dsDNA molecule. The invention also provides methods of synthesizing DNA molecules by assembling oligonucleotide members of a library that contains less than 20,000 members that can be assembled into all possible DNA sequences.

Classes IPC  ?

• C12N 15/11 - Fragments d'ADN ou d'ARNLeurs formes modifiées
• C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
• C40B 40/06 - Bibliothèques comprenant des nucléotides ou des polynucléotides ou leurs dérivés

Abrégé

The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.

Classes IPC  ?

• C12N 15/09 - Technologie d'ADN recombinant
• C12N 15/11 - Fragments d'ADN ou d'ARNLeurs formes modifiées
• C12P 19/34 - Polynucléotides, p. ex. acides nucléiques, oligoribonucléotides
• C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques