Methods for testing oligonucleotide arrays
First Claim
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1. A method of manufacturing oligonucleotide arrays comprising manufacturing oligonucleotide arrays by spatially directed oligonucleotide synthesis in high volume and testing arrays selected from among the high volume manufactured nucleic acid probe arrays.
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Abstract
Methods for testing oligonucleotide arrays are disclosed including methods for testing the efficiency of nucleotide coupling; methods for testing amounts of deprotected oligonucleotides; methods for determining amounts of depurinated oligonucleotides; and methods of detecting the presence of cleavable structural features, such as double-stranded nucleic acids.
53 Citations
54 Claims
- 1. A method of manufacturing oligonucleotide arrays comprising manufacturing oligonucleotide arrays by spatially directed oligonucleotide synthesis in high volume and testing arrays selected from among the high volume manufactured nucleic acid probe arrays.
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7. A testing method comprising:
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providing a substrate having a surface with linkers having active sites for oligonucleotide synthesis;
synthesizing an ensemble of sequence-specific oligonucleotides on the substrate by spatially directed oligonucleotide synthesis;
exposing an area of the substrate to a test condition; and
determining the amount of oligonucleotides having a structural feature. - View Dependent Claims (8, 9, 10, 11, 12, 13)
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14. A method for testing the efficiency of nucleotide coupling in the synthesis of an oligonucleotide array by spatially directed oligonucleotide synthesis comprising the steps of:
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providing a substrate having a surface having linkers with active sites;
coupling first protected nucleotides to the active sites of the linkers in a first area and at least one second area of the substrate and capping uncoupled, unprotected active sites in said first and second areas of the substrate, deprotecting protected nucleotides in the second area(s), coupling second protected nucleotides to deprotected nucleotides in the second area(s) and capping uncoupled, unprotected nucleotide in the second area(s);
optionally repeating the previous step in at least one area of the substrate and capping uncoupled, unprotected nucleotides and active sites of the linkers in said area(s);
determining the amount of uncapped nucleotide in at least two areas; and
comparing the amounts determined, wherein the comparative amount indicates the efficiency of nucleotide coupling of the two areas. - View Dependent Claims (15, 16, 17, 18)
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19. A method for testing the efficiency of nucleotide coupling in the synthesis of an oligonucleotide array by spatially directed oligonucleotide synthesis comprising the steps of:
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providing a substrate having a surface having cleavable linkers, wherein the linkers comprise detectable labels that are releasable upon cleavage of the linkers and active sites for nucleotide coupling;
coupling at least one nucleotide to the active sites whereby the detectable labels are coupled to the nucleotide, thereby producing a detectably labeled coupled nucleotide;
capping uncoupled active sites after at least one coupling step;
cleaving the cleavable linkers to release the detectably labeled coupled nucleotides; and
determining the number of nucleotides coupled to the cleaved linker, wherein the number of nucleotides coupled to a cleaved linker indicates the efficiency of nucleotide coupling. - View Dependent Claims (20)
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- 21. A quality control process for manufacturing nucleic acid probe arrays comprising the steps of manufacturing nucleic acid probe arrays on a substrate having a surface in high volume and testing one or more arrays selected from among the high volume manufactured nucleic acid probe arrays.
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46. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate comprising the steps of:
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(a) providing a substrate having a surface with linkers having an active site for oligonucleotide synthesis, the linkers being resistant to cleavage under cleavage conditions;
(b) synthesizing oligonucleotides on said linkers having an active site for oligonucleotide synthesis in an area of the substrate, said oligonucleotides having one or more active sites for attaching a detectable label;
(c) attaching a detectable label to the oligonucleotides; and
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(d) determining the amount of detectable label in the area of substrate, said amount of detectable label being a determination of said amount of depurination.
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47. A quality control process for nucleic acid arrays comprising:
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(a) manufacturing a nucleic acid array comprising the steps of;
(i) providing a substrate having a surface for nucleic acid deposition, wherein the substrate is selected from the group consisting of glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, silica, (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, and combinations thereof; and
(ii) placing an ensemble of different nucleic acids on the surface of the substrate, thereby manufacturing a nucleic acid array;
(b) manufacturing nucleic acid arrays in high volume by repeating steps (a)(i) and (a)(ii); and
(c) testing selected arrays of the high volume manufactured. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54)
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Specification