METHODS FOR RAPID PRODUCTION OF TARGET DOUBLE-STRANDED DNA SEQUENCES
First Claim
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1. A method for generating a double-stranded DNA sequence, the method comprising the following steps in the order set forth below:
- (a) synthesizing in parallel a plurality of single-stranded oligonucleotide primary constructs, each primary construct comprising(i) an internal region, wherein a plurality of internal regions define the DNA sequence and wherein each internal region has at least one portion overlapping and complementary to a portion of an internal region of at least one different primary construct, and(ii) a 5′ and
a 3′
flanking region flanking the 5′ and
the 3′
end of the internal region, each of the flanking regions including both a primer recognition site and a recognition site for a restriction enzyme to cut the primary construct at each of the junctions of said internal region and said flanking regions;
(b) selectively amplifying at least a subset of the plurality of the single-stranded primary constructs of step (a) to generate multiple copies by conducting a polymerase chain reaction (PCR) process on said single-stranded primary constructs using a set of PCR primers that target the primer recognition site(s) in said flanking regions to create at least one amplified pool of double-stranded primary constructs;
(c) removing said flanking regions, each of which includes the primer recognition site and the restriction enzyme recognition site, from the amplified double-stranded primary constructs of step (b) by digesting said constructs with the restriction enzyme, thereby generating a plurality of double-stranded internal regions; and
(d) conducting repetitive denaturing, annealing and extension procedures on the internal regions of step (c) in the presence of a polymerase and dNTPs, but without the presence of primers so that no amplification occurs, to allow the overlapping internal regions to hybridize and assemble, thereby generating said double-stranded DNA sequence.
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Abstract
It has been previously disclosed that DNA segments can be made in massively parallel chemical synthesis operations on a common substrate followed by release of the segments from the substrate and assembly of the segments into target DNA molecules. Here it is taught that if the DNA primary constructs are sufficiently long and properly designed, that the copy numbers of the primary constructs can be multiplied as needed by a PCR process using as a template regions at the ends of the primary constructs. The end regions, called flanking regions, can also be designed so that they may be cleaved easily from the amplification products. The target double-stranded DNA can then be assembled from the cleaved fragments. Hundreds of thousands of oligonucleotides can be synthesized and assembled into many different individual genes by this process in a relatively quick and efficient process.
26 Citations
22 Claims
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1. A method for generating a double-stranded DNA sequence, the method comprising the following steps in the order set forth below:
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(a) synthesizing in parallel a plurality of single-stranded oligonucleotide primary constructs, each primary construct comprising (i) an internal region, wherein a plurality of internal regions define the DNA sequence and wherein each internal region has at least one portion overlapping and complementary to a portion of an internal region of at least one different primary construct, and (ii) a 5′ and
a 3′
flanking region flanking the 5′ and
the 3′
end of the internal region, each of the flanking regions including both a primer recognition site and a recognition site for a restriction enzyme to cut the primary construct at each of the junctions of said internal region and said flanking regions;(b) selectively amplifying at least a subset of the plurality of the single-stranded primary constructs of step (a) to generate multiple copies by conducting a polymerase chain reaction (PCR) process on said single-stranded primary constructs using a set of PCR primers that target the primer recognition site(s) in said flanking regions to create at least one amplified pool of double-stranded primary constructs; (c) removing said flanking regions, each of which includes the primer recognition site and the restriction enzyme recognition site, from the amplified double-stranded primary constructs of step (b) by digesting said constructs with the restriction enzyme, thereby generating a plurality of double-stranded internal regions; and (d) conducting repetitive denaturing, annealing and extension procedures on the internal regions of step (c) in the presence of a polymerase and dNTPs, but without the presence of primers so that no amplification occurs, to allow the overlapping internal regions to hybridize and assemble, thereby generating said double-stranded DNA sequence. - View Dependent Claims (2, 3, 4, 5, 6, 8, 17, 18, 22)
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7. (canceled)
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9. A method for making a target doubled-stranded DNA sequence, the method comprising the following steps in the order set forth below:
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(a) synthesizing on a microarray a plurality of single stranded oligonucleotide primary constructs, each primary construct being at least 70 nucleotides in length and including an internal region of at least 40 nucleotides, wherein—
a plurality of internal regions define the DNA sequence and wherein each internal region has at least one portion overlapping and complementary to a portion of an internal region of at least one different primary construct, and a 5′ and
3′
flanking region flanking the 5′ and
3′
end of said internal region, each of the flanking regions being a region of at least 15 nucleotides and including a primer recognition site and including a recognition site for a restriction enzyme at each of the junctions of said internal region and said flanking regions;(b) selectively amplifying at least a subset of the single-stranded primary constructs by conducting a polymerase chain reaction (PCR) process on said single-stranded primary constructs of step (a) using a set of PCR primers that target the primer recognition site(s) in said flanking regions to create at least one amplified pool of double-stranded primary constructs; (c) removing said flanking regions, each of which includes the primer recognition site and the restriction enzyme recognition site . . . from the amplified double-stranded primary constructs of step (b) by digesting said constructs with the restriction enzyme, thereby generating a plurality of double-stranded internal regions; and (d) conducting repetitive denaturing, annealing and extension procedures on the internal regions of step (c) in the presence of a polymerase and dNTPs, but without the presence of primers so that no amplification occurs to allow overlapping internal regions to hybridize and assemble thereby generating said double-stranded DNA sequence. - View Dependent Claims (10, 11, 12, 13, 14, 16, 19, 20)
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15. (canceled)
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21. A method of generating a double-stranded DNA sequence, the method comprising the following steps in the order set forth below:
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(a) synthesizing in parallel a plurality of single stranded oligonucleotide primary constructs, each primary construct comprising a 5′
-flanking region, an internal region, and a 3′
flanking region, wherein(i) a plurality of the internal regions define the DNA sequence and wherein each single stranded internal region has a sequence complementary to the next internal region, (ii) each of the two flanking regions includes a primer recognition site and a recognition site for a restriction enzyme at each of the junctions of the internal region and flanking region; (b) selectively amplifying at least a subset of the plurality of the single-stranded primary constructs of step (a) to generate multiple copies by conducting a polymerase chain reaction (PCR) process on said single-stranded primary constructs using a set of PCR primers that target the primer recognition site(s) in said flanking regions to create at least one amplified pool of double-stranded primary constructs; (c) removing said flanking regions, each of which includes the primer recognition site and the restriction enzyme recognition site, from the amplified double-stranded primary constructs of step (b) by digesting said constructs with the restriction enzyme thereby generating a plurality of double-stranded internal regions, wherein the enzyme is a type II restriction enzyme that makes a blunt end after cleavage of the DNA sequence; and (d) conducting repetitive denaturing, annealing and extension procedures on the internal regions of step (c) in the presence of a polymerase and dNTPs, but without the presence of primers so that no amplification occurs, se-as to allow the overlapping internal regions to hybridize and assemble by extension, thereby generating said double-stranded DNA sequence.
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Specification
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Current AssigneeWisconsin Alumni Research Foundation (University of Wisconsin System)
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Original AssigneeWisconsin Alumni Research Foundation (University of Wisconsin System)
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InventorsRodesch, Matt J., Richmond, Kathryn E., Sussman, Michael R.
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Application NumberUS11/148,401Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current506/26CPC Class CodesC12Q 1/686 Polymerase chain reaction [...C12Q 2533/107 Probe or oligonucleotide li...
