Parallel preparation of high fidelity probes in an array format

A method of purifying a set of oligonucleotides, comprising the steps of

• a) providing a solid substrate comprising a plurality of ribonucleotides attached thereto at a density of 200 to 2000 pmoles per cm2, one said ribonucleotide shown below
• wherein PG1 is protecting group 1, PG2 is an alkaline resistant protecting group, B is a naturally or non-naturally occurring base, and said ribonucleotide is attached to said substrate through the 5′-hydroxyl group;
• b) selectively removing PG1 in pre-selected areas to provide a plurality of free 3′-hydroxyl groups on said ribonucloetide;
• c) reacting said free 3′-hydroxyl groups with a 2′-deoxyribonucleotide having the structure
• wherein PG3 is DMT, B is a naturally or non-naturally occurring base in which the exocyclic amine groups are protected with alkaline labile protecting groups, and RG is a reactive group to couple said 2′-deoxyribonucleotide to said ribonucleotide to provide the structure
• d) selectively removing PG3 from the 5′-hydroxyl of said 2′-deoxyribonucleotide in pre-selected areas to provide a plurality of free 5′-hydroxyl groups;
• e) reacting said free 5′-hydroxyl groups with an additional 2′-deoxyribonucleotide having the structure
• wherein PG4 is DMT, to yield a product of the structure
• f) repeating steps d and e one or more times to provide said oligonucleotides attached to said solid substrate;
• g) deprotecting said set of oligonucleotides while said oligonucleotides are still attached to said substrate by subjecting said oligonucleotides to alkaline conditions, wherein said alkaline conditions remove said alkaline labile protecting groups acting to protect said exocyclic amines and in addition cleave depurinated DNA, leaving a 3′-end of the cleaved depurinated strand attached to the substrate and releasing a truncated fragment;
• h) washing the solid support to remove said released truncated fragments and protecting groups, leaving full length oligonucleotides having a DMT group on the 5′-hydroxyl group and truncated oligonucleotides without the 5′-DMT group;
• i) removing PG2 from one or more of said ribonucleotides to provide a free 2′-hydroxyl group on each of said one or more ribonucleotides;
• j) transesterifying each of said one or more ribonucleotides to yield said solid substrate having a cyclic ester attached thereto and a mixture of full length oligonucleotides having 5′-DMT groups and free 3′-hydroxyl groups and having the structure
• and truncated fragments lacking the DMT group;
• k) applying the mixture to hydrophobic oligonucleotide purification resin to isolate only those oligonucleotides having the 5′-DMT group to yield full length oligonucleotides;
• and
• l) removing the 5′-DMT group to provide sets of full length oligonucleotides having both 5′- and 3′-hydroxyl groups.