Array oligomer synthesis and use
First Claim
1. A method for parallel synthesis of an array of selected multimers on a substrate comprising isolated reaction sites containing one or more protected initiating moieties, the method comprising:
- (a) selectively irradiating isolated reaction sites to generate deprotected initiating moieties at the irradiated isolated reaction sites;
(b) coupling one or more monomers to the deprotected initiating moieties;
(c) repeating steps (a)-(b) until the array of selected multimers has been synthesized;
wherein the multimers synthesized comprise multimers from about 75 to 200 monomers is length.
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Abstract
The present disclosure provides efficient and reproducible methods for individually synthesizing oligomers in a parallel manner (e.g., oligonucleotides) on a solid support to produce pools of oligomers. Pools of oligonucleotides can be used for a variety of genomic and proteomic applications, including synthesis of genes or long DNA of any arbitrary sequence, PCR template amplification, and to generate primers for multiplexing PCR or transcription. Rapid availability of these oligonucleotide products will greatly accelerate the processes of de novo protein design, vaccine development, production of short RNA fragments, such as siRNA, oligonucleotide-based drug screening, and SNP sample preparation.
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Citations
37 Claims
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1. A method for parallel synthesis of an array of selected multimers on a substrate comprising isolated reaction sites containing one or more protected initiating moieties, the method comprising:
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(a) selectively irradiating isolated reaction sites to generate deprotected initiating moieties at the irradiated isolated reaction sites;
(b) coupling one or more monomers to the deprotected initiating moieties;
(c) repeating steps (a)-(b) until the array of selected multimers has been synthesized;
wherein the multimers synthesized comprise multimers from about 75 to 200 monomers is length. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14)
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10. The method of claim 10, wherein the photo-reagent precursors are selected from the group consisting of acid precursors and base precursors.
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15. A method of generating a DNA sequence comprising:
- selecting suitable oligonucleotide subchains for the assembly of the DNA sequence, wherein the subchains are designed so that the DNA sequence is formed by the annealed subchains;
parallel synthesis of the subchains on a solid support, wherein the subchains are from about 75 to about 150 nucleotides in length;
annealing the subchains;
ligating the annealed subchains to generate the DNA sequence. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
- selecting suitable oligonucleotide subchains for the assembly of the DNA sequence, wherein the subchains are designed so that the DNA sequence is formed by the annealed subchains;
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27. A method of generating a DNA sequence comprising:
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a) selecting suitable oligonucleotide subchains for the assembly of tie DNA sequence, wherein the subchains are designed so that the duplex DNA sequence is formed by the annealed subchains;
b) parallel synthesis of the subchains on a solid support, wherein a 98% coupling efficiency or greater per step of oligonucleotide synthesis is achieved;
c) annealing the subchains;
d) ligating the annealed subchains to generate the DNA sequence.
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28. A method of generating a library of short RNA molecules comprising:
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a) synthesizing an array of selected oligonucleotides on a substrate, wherein the selected oligonucleotides comprise an RNA polymerase promoter sequence, wherein the substrate comprises protected initiating moieties at specific reaction sites on the substrate, comprising;
i) contacting the substrate with a liquid solution comprising one or more photo-reagent precursors, such that the liquid solution is in contact with the protected initiating moieties;
ii) isolating the specific reaction sites;
iii) selectively irradiating isolated reaction sites to produce one or more photo-generated reagents, wherein the photo-generated reagents are effective to deprotect the initiating moieties at the irradiated reaction sites;
iv) contacting the substrate with a monomer, wherein the monomer comprises an unprotected reactive site and a protected reactive site, under conditions such that the unprotected reactive site of the monomer couples with the deprotected initiating moieties so as to create an attached monomer and protected initiating moieties;
v) repeating steps (i)-(iv) until the array of selected oligonucleotides has been synthesized;
wherein the selected oligonucleotides comprise two specific primer sequences for DNA amplification;
b) cleaving of the selected oligonucleotides from the solid support;
c) amplifying the selected oligonucleotides using primers that recognize the specific primer sequences, wherein double stranded DNA comprising the sequences of the selected oligonucleotides is generated;
d) in vitro transcription of the amplified double stranded DNA using an RNA polymerase that recognizes the RNA promoter sequence, wherein a library of short RNA molecules is generated. - View Dependent Claims (29, 30, 32, 33)
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31. The method of claim 31, wherein the selected oligonucleotides are cleaved from the solid support using RNase A.
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34. A method of large-scale Single Nucleotide Polymorphism (SNP) detection in a DNA sample comprising:
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a) designing an array of primer pairs that will amplify an array of amplicons from the DNA sample, wherein each amplicon comprises one or more SNPs;
b) synthesizing the array of primer pairs on a substrate, wherein the substrate comprises protected initiating moieties at specific reaction sites on the substrate, comprising;
i) contacting the substrate with a liquid solution comprising one or more photo-reagent precursors, such that the liquid solution is in contact with the protected initiating moieties;
ii) isolating the specific reaction sites;
iii) selectively irradiating isolated reaction sites to produce one or more photo-generated reagents, wherein the photo-generated reagents are effective to deprotect the initiating moieties at the irradiated reaction sites;
iv) contacting the substrate with a monomer, wherein the monomer comprising an unprotected reactive site and a protected reactive site, under conditions such that the unprotected reactive site of the monomer couples with the deprotected initiating moieties so as to create an attached monomer and protected initiating moieties;
v) repeating steps (i)-(iv) until the array of selected oligonucleotides has been synthesized;
wherein a single primer pair is synthesized in each reaction site on the substrate;
b) DNA amplification of the amplicons using the primer pairs, wherein a single amplicon is generated in each reaction site on the substrate;
c) detection of the one or more SNPs present in each amplicon. - View Dependent Claims (35)
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36. A method of large-scale Single Nucleotide Polymorphism (SNP) detection in a DNA sample comprising:
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a) designing an array of primer pairs that will amplify an array of amplicons from the DNA sample, wherein each primer pair will only amplify an amplicon if a particular SNP is present in the DNA sample;
b) synthesizing the array of primer pairs on a substrate, wherein the substrate comprises protected initiating moieties at specific reaction sites on the substrate, comprising;
i) contacting the substrate with a liquid solution comprising one or more photo-reagent precursors, such that the liquid solution is in contact with the protected initiating moieties;
ii) isolating the specific reaction sites;
iii) selectively irradiating isolated reaction sites to produce one or more photo-generated reagents, wherein the photo-generated reagents are effective to deprotect the initiating moieties at the irradiated reaction sites;
iv) contacting the substrate with a monomer, wherein the monomer comprising an unprotected reactive site and a protected reactive site, under conditions such that the unprotected reactive site of the monomer couples with the deprotected initiating moieties so as to create an attached monomer and protected initiating moieties;
v) repeating steps (i)-(iv) until the array of selected oligonucleotides has been synthesized;
wherein a single primer pair is synthesized in each reaction site on the substrate;
b) DNA amplification of the amplicons using the primer pairs, wherein the amplification of an amplicon indicates the presence of a particular SNP in the DNA sample.
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37. A method of generating an oligonucleotide library comprising:
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a) synthesizing an array of selected oligonucleotides on a substrate, wherein the selected oligonucleotides comprise two specific primer sequences and a variable region of sequence, wherein the substrate comprises protected initiating moieties at specific reaction sites on the substrate, comprising;
i) contacting the substrate with a liquid solution comprising one or more photo-reagent precursors, such that the liquid solution is in contact with the protected initiating moieties;
ii) isolating the specific reaction sites;
iii) selectively irradiating isolated reaction sites to produce one or more photo-generated reagents, wherein the photo-generated reagents are effective to deprotect the initiating moieties at the irradiated reaction sites;
iv) contacting the substrate with a monomer, wherein the monomer comprising an unprotected reactive site and a protected reactive site, under conditions such that the unprotected reactive site of the monomer couples with the deprotected initiating moieties so as to create an attached monomer and protected initiating moieties;
v) repeating steps (i)-(iv) until the array of selected oligonucleotides has been synthesized;
b) cleavage of the selected oligonucleotides from the solid support;
c) DNA amplification of the selected oligonucleotides using primers that recognize the specific primer sequences, thereby generating an oligonucleotide library of double stranded DNA sequences comprising the variable region sequences of the selected oligonucleotides.
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Specification