US20130059296A1 - Compositions and Methods For High Fidelity Assembly of Nucleic Acids - Google Patents
Compositions and Methods For High Fidelity Assembly of Nucleic Acids Download PDFInfo
- Publication number
- US20130059296A1 US20130059296A1 US13/592,827 US201213592827A US2013059296A1 US 20130059296 A1 US20130059296 A1 US 20130059296A1 US 201213592827 A US201213592827 A US 201213592827A US 2013059296 A1 US2013059296 A1 US 2013059296A1
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- nucleic acid
- cohesive
- acid fragments
- oligonucleotides
- end double
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B30/20—Sequence assembly
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
- C12Q2521/301—Endonuclease
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- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/50—Other enzymatic activities
- C12Q2521/501—Ligase
Definitions
- nucleic acids are made (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest.
- nucleic acids are made (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest.
- multiplex assembly techniques are being developed for assembling oligonucleotides into larger synthetic nucleic acids that can be used in research, industry, agriculture, and/or medicine.
- one limitation of currently available assembly techniques is the relatively high error rate. As such, high fidelity, low cost assembly methods are needed.
- the plurality of blunt-end double-stranded nucleic acid fragments can be provided by releasing a plurality of oligonucleotides synthesized on a solid support, and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.
- oligonucleotides synthesized therefrom are chemically, enzymatically, or physically cleaved or otherwise released from the microarrays for further amplification, restriction enzyme digestion and/or assembly.
- two or more pairs of complementary cohesive ends between different nucleic acid fragments may be designed or selected to have identical or similar sequences in order to promote the assembly of products containing a relatively random arrangement (and/or number) of the fragments that have similar or identical cohesive ends. This may be useful to generate libraries of nucleic acid products with different sequence arrangements and/or different copy numbers of certain internal sequence regions.
- each of the two pools comprises a plurality of nucleic acid fragments, each nucleic acid fragment of the first pool having a terminal end complementary to a terminal end of a nucleic acid fragment in the second pool.
- the at least two pools can be formed by splitting the population of oligonucleotides into the at least two pools and amplifying the oligonucleotides in each pool separately. In other embodiments, the at least two pools can be formed by releasing (e.g.
- one or more steps of an amplification and/or assembly reaction may be automated using one or more automated sample handling devices (e.g., one or more automated liquid or fluid handling devices).
- Automated devices and procedures may be used to deliver reaction reagents, including one or more of the following: starting nucleic acids, buffers, enzymes (e.g., one or more ligases and/or polymerases), nucleotides, salts, and any other suitable agents such as stabilizing agents.
- Automated devices and procedures also may be used to control the reaction conditions. For example, an automated thermal cycler may be used to control reaction temperatures and any temperature cycles that may be used.
- Assembly reaction mixtures may be transferred from one component of the system to another using automated devices and procedures (e.g., robotic manipulation and/or transfer of samples and/or sample containers, including automated pipetting devices, micro-systems, etc.).
- automated devices and procedures e.g., robotic manipulation and/or transfer of samples and/or sample containers, including automated pipetting devices, micro-systems, etc.
- the system and any components thereof may be controlled by a control system.
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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JP2014527291A JP2014526899A (ja) | 2011-08-26 | 2012-08-23 | 核酸の高忠実度アセンブリのための組成物および方法 |
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IL302248A IL302248A (en) | 2011-08-26 | 2012-08-23 | Preparations and methods for high-fidelity assembly of nucleic acids |
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US18/324,339 US20230407316A1 (en) | 2011-08-26 | 2023-05-26 | Compositions and methods for high fidelity assembly of nucleic acids |
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