Method for the synthesis of DNA sequences
First Claim
1. A method for the fabrication of a DNA construct of a target sequence comprising the steps of(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate would make up the target DNA sequence, wherein the single stranded segments overlap in the regions where the segments are complementary to each other;
- (b) making a DNA microarray on a substrate, the microarray made up of features having single stranded DNA molecules, the single stranded DNA molecules selected to be overlapping sequence segments of the target sequence, the single stranded DNA molecules being made attached by linkers to the substrate, the linkers being stable under the conditions of DNA synthesis for the microarray, the linkers also being capable of being rendered photo-labile by chemical treatment;
(c) detaching the single stranded DNA molecules from the substrate by first applying a chemical modification to the linkers to render the linkers light labile and then by selectively applying light to cleave the labile linkers in areas of single stranded DNA molecules thus detaching the single stranded DNA molecules from the substrate; and
(d) allowing the single stranded DNA molecules detached in step (c) to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′
overhang contained on either strand.
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Abstract
A method is disclosed for the direct synthesis of double stranded DNA molecules of a variety of sizes and with any desired sequence. The DNA molecule to be synthesis is logically broken up into smaller overlapping DNA segments. A maskless microarray synthesizer is used to make a DNA microarray on a substrate in which each element or feature of the array is populated by DNA of a one of the overlapping DNA segments. The DNA segments are released from the substrate and held under conditions favoring hybridization of DNA, under which conditions the segments will spontaneously hybridize together to form the desired DNA construct. This method makes possible the remote assembly of DNA sequence, through a process analogous to facsimile transmission of documents, since the information on DNA to be made can be transmitted remotely to an instrument which can then synthesize any needed DNA sequence from the information.
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Citations
9 Claims
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1. A method for the fabrication of a DNA construct of a target sequence comprising the steps of
(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate would make up the target DNA sequence, wherein the single stranded segments overlap in the regions where the segments are complementary to each other; -
(b) making a DNA microarray on a substrate, the microarray made up of features having single stranded DNA molecules, the single stranded DNA molecules selected to be overlapping sequence segments of the target sequence, the single stranded DNA molecules being made attached by linkers to the substrate, the linkers being stable under the conditions of DNA synthesis for the microarray, the linkers also being capable of being rendered photo-labile by chemical treatment; (c) detaching the single stranded DNA molecules from the substrate by first applying a chemical modification to the linkers to render the linkers light labile and then by selectively applying light to cleave the labile linkers in areas of single stranded DNA molecules thus detaching the single stranded DNA molecules from the substrate; and (d) allowing the single stranded DNA molecules detached in step (c) to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′
overhang contained on either strand. - View Dependent Claims (2, 3, 4, 5, 7)
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6. A method for the fabrication of a target DNA construct of comprising the steps of
(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate make up the target DNA construct, wherein the single stranded segments overlap in the regions where the segments are complementary to each other; -
(b) making the single stranded DNA molecules in an array on a substrate, the single stranded DNA molecules linked to the substrate by base labile linkers; (c) releasing the single stranded DNA molecules from the substrate by alkaline treatment of the array to cause the base labile linkers to release the single stranded DNA molecules; and (d) allowing the single stranded DNA molecules to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′
overhang contained on either strand. - View Dependent Claims (8)
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9. A method for the fabrication of a DNA construct of a target sequence comprising the steps of
(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate would make up the target DNA sequence, wherein the single stranded segments overlap in the regions where the segments are complementary to each other; -
(b) making a DNA microarray on a substrate in which single stranded DNA molecules constructed in the microarray are the overlapping sequence segments, the DNA molecules each being attached to the substrate by a linker which is stable while the microarray is being made, the linker being capable of being rendered photo-labile by chemical treatment; (c) releasing the single stranded DNA molecules from the substrate by chemical treatment to modify the linkers to become light labile and then using light to cleave the linkers to release the single stranded DNA molecules; (d) allowing the single stranded DNA molecules released in step (c) to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′
overhang contained on either strand; and(e) ligating said assembled DNA molecules into a DNA molecule of the target sequence.
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Specification