Claims
- 1. A method for manufacturing oligonucleotide arrays comprising manufacturing oligonucleotide arrays in high volume by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction, and testing arrays selected from among the high volume of manufactured oligonucleotide arrays.
- 2. The method of claim 1, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 3. The method of claim 1, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 4. A method for manufacturing oligonucleotide arrays comprising manufacturing oligonucleotide arrays in high volume by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction, and testing selected arrays for the amount of depurination of oligonucleotides.
- 5. The method of claim 4, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 6. The method of claim 4, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 7. A quality control process for manufacturing oligonucleotide arrays comprising manufacturing oligonucleotide arrays in high volume by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction, and testing arrays selected from among the high volume of manufactured oligonucleotide arrays.
- 8. The method of claim 7, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 9. The method of claim 7, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 10. A quality control process for manufacturing oligonucleotide arrays comprising the steps of:
a) manufacturing oligonucleotide arrays in high volume on a substrate having a surface in a 5′ to 3′ or a 3′ to 5′ direction; and b) testing one or more arrays selected from among the high volume of manufactured oligonucleotide arrays.
- 11. The method of claim 10, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 12. The method of claim 10, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 13. The method of claim 10, 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.
- 14. A method for testing the efficiency of nucleotide coupling in the synthesis of an oligonucleotide array by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
a) providing a substrate having a surface with linkers having active sites; b) coupling first protected nucleotides to active sites 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; c) deprotecting protected nucleotides in the second area(s), coupling second protected nucleotides to deprotected nucleotides in the second area(s) and capping uncoupled, unprotected nucleotides in the second area(s); d) optionally repeating step (c) in at least one area of the substrate and capping uncoupled, unprotected active sites of the linkers in said area(s); e) determining the amount of uncapped nucleotides in at least two areas; and f) comparing the determined amounts, wherein the comparative amount indicates the efficiency of nucleotide coupling of the two areas.
- 15. The method of claim 14, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 16. The method of claim 14, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 17. A method for testing the efficiency of nucleotide coupling in the synthesis of an oligonucleotide array by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
a) providing a substrate having a surface with cleavable linkers, wherein the linkers comprise detectable labels that are releasable upon cleavage of the linkers and active sites for nucleotide coupling; b) 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; c) capping uncoupled active sites after at least one coupling step; d) cleaving the cleavable linkers to release the detectably labeled coupled nucleotides; and e) 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.
- 18. The method of claim 17, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 19. The method of claim 17, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 20. A method for comparing the relative efficiency of two test conditions to cause deprotection of oligonucleotides synthesized on a substrate by spatially directed nucleic acid synthesis in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
a) providing a substrate on which an ensemble of oligonucleotides has been synthesized in a 5′ to 3′ or a 3′ to 5′ direction, wherein an active site on a free terminal nucleotide of the oligonucleotides bears a protecting group; b) exposing a first area of the substrate to a first test condition; c) exposing a second area of the substrate to a second test condition; and d) determining the amount of unprotected active sites in the first and second areas, whereby the relative amount indicates the relative efficiency of the test conditions to cause deprotection.
- 21. The method of claim 20, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 22. The method of claim 20, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 23. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
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 in a 5′ to 3′ or a 3′ to 5′ direction 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 nucleotides; and d) determining the amount of detectable label in the area of the substrate, wherein said amount of detectable label determines the amount of depurination.
- 24. The method of claim 23, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 25. The method of claim 23, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 26. The method of claim 23, wherein the oligonucleotides synthesized on a substrate are synthesized by spatially directed oligonucleotide synthesis.
- 27. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
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 an ensemble of sequence-specific oligonucleotides in a 5′ to 3′ or 3′ to 5′ direction in an area of the substrate, the oligonucleotides having active sites for attaching a detectable label; c) attaching a detectable label to the oligonucleotides in the ensemble; d) exposing the ensemble to a test condition; e) exposing the ensemble to cleavage conditions that cause cleavage of depurinated oligonucleotides; and f) determining the amount of detectable label in the area, wherein said amount of detectable label is a determination of the amount of depurination.
- 28. The method of claim 27, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 29. The method of claim 27, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 30. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction comprising the steps of:
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 an ensemble of sequence-specific oligonucleotides in a 5′ to 3′ or a 3′ to 5′ direction in an area of the substrate under a test condition, the oligonucleotides having active sites for attaching a detectable label; c) attaching a detectable label to the active sites; d) exposing the ensemble to cleavage conditions that cause cleavage of depurinated oligonucleotides; and e) determining the amount of detectable label in the area, wherein said amount of detectable label is a determination of the amount of depurination.
- 31. The method of claim 30, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 32. The method of claim 30, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 33. A method for determining whether an ensemble of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction contains double-stranded nucleic acids formed within an oligonucleotide or between oligonucleotides in an ensemble comprising the steps of:
a) providing a substrate on which an ensemble of sequence-specific oligonucleotides has been synthesized in a 5′ to 3′ or a 3′ to 5′ direction in an area of the substrate, wherein the oligonucleotides in the ensemble are attached to a detectable label that is released from the area upon cleavage of the oligonucleotides; b) contacting the ensemble with an agent that cleaves double-stranded nucleic acids, whereby cleavage of oligonucleotides forming double-stranded nucleic acids releases detectable label from the area; and c) determining the amount of detectable label remaining in the area, wherein the amount of remaining detectable label is inversely related to the amount of double-stranded nucleic acids.
- 34. The method of claim 33, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 35. The method of claim 33, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 36. A testing method comprising the steps of:
a) providing a substrate having a surface with linkers having active sites for oligonucleotide synthesis; b) synthesizing an ensemble of sequence-specific oligonucleotides on the linkers on the substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ or a 3′ to 5′ direction; c) exposing an area of the substrate to a test condition; and d) determining the amount of oligonucleotides having a structural feature.
- 37. The method of claim 36, wherein the oligonucleotide synthesis is performed in a 3′ to 5′ direction and comprises using a 5′-protected 3′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 38. The method of claim 36, wherein the oligonucleotide synthesis is performed in a 5′ to 3′ direction and comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
- 39. The method of claim 36, further comprising exposing a second area of the substrate to a second test condition and determining the relative amount of oligonucleotides having a structural feature in the areas exposed to the test conditions, whereby the relative amount indicates the relative efficiency of the test conditions to cause the appearance of the structural feature.
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser. No. 09/781,537, filed Feb. 8, 2001, which is a continuation of U.S. application Ser. No. 08/995,265, filed Dec. 19, 1997, which is a continuation of U.S. application Ser. No. 08/531,155, filed Sep. 18, 1995.
[0002] The entire teachings of the above applications are incorporated herein by reference.
Continuations (3)
|
Number |
Date |
Country |
Parent |
09781537 |
Feb 2001 |
US |
Child |
10457994 |
Jun 2003 |
US |
Parent |
08995265 |
Dec 1997 |
US |
Child |
09781537 |
Feb 2001 |
US |
Parent |
08531155 |
Sep 1995 |
US |
Child |
08995265 |
Dec 1997 |
US |