Methods for testing oligonucleotide arrays
View Patent ↗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.
1. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ 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′ 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.
2. The method of claim 1 , wherein the oligonucleotide synthesis comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.
3. A method for determining the amount of depurination of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis in a 5′ to 3′ 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′ 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.
4. The method of claim 3 , wherein the oligonucleotide synthesis comprises using a 3′-protected 5′-O-phosphoramidite-activated deoxynucleoside in the oligonucleotide synthesis.