Parallel preparation of high fidelity probes in an array format
View Patent ↗The present invention provides massively parallel oligonucleotide synthesis and purification for applications that utilize large collections of defined high-fidelity oligonucleotides (e.g., from about 10 1 to about 10 5 different sequences, generally between 25-160 bases in length).
1. A method for massively parallel oligonucleotide probe synthesis and release of the synthesized oligonucleotides from an array of probes on a solid substrate, the method comprising:
providing a solid substrate;
attaching a plurality of linkers to the substrate, each linker comprising a cleavable moiety, wherein the cleavable moiety is activatable only at a distinct set of conditions and wherein activation of the cleavable moiety disrupts the linker to allow release of the synthesized oligonucleotides and to provide a substrate with a plurality of attached linkers, wherein the linker has the structure shown below
and wherein PG 1 is protecting group 1, PG 2 is protecting group 2, B is a naturally or non-naturally occurring base, and the linker is attached to the substrate through the 5′-hydroxyl group;
attaching a first monomer to at least one of the plurality of attached linkers to provide an attached first monomer;
attaching a second monomer to a least one of the attached first monomers or the plurality of attached linkers to provide an attached second monomer;
attaching a third monomer to a least one of the attached first monomer, the attached second monomer or the plurality of attached linkers to provide an attached third monomer;
repeating the steps of attaching monomers until the desired array of synthesized oligonucleotides is complete; and
subjecting the array to the distinct set of conditions to activate the cleavable moieties, wherein activation comprises removal of PG 2 , wherein removal of PG 2 allows base-induced intramolecular transesterification, and wherein transesterification releases the synthesized oligonucleotides from the array.
2. The method of claim 1 , wherein the transesterification produces released synthesized oligonucleotides with 3′ hydroxyl groups.
3. The method of claim 1 , wherein the distinct set of conditions comprises an elevated pH level.
4. The method of claim 3 , wherein the elevated pH level is from about 9 to about 12.
5. The method of claim 1 , wherein the distinct set of conditions comprises addition of metal ions.
6. The method of claim 3 , wherein the distinct set of conditions further comprises addition of metal ions.
7. The method of claim 1 , wherein PG 1 is DMT.
8. The method of claim 7 , wherein PG 1 is removed for attachment of the first monomer by exposure to an acid generated by a photoacid generator in the presence of electromagnetic radiation of an appropriate wavelength and in the presence of an acid scavenger.
9. The method of claim 1 , wherein PG 2 is selected from the group consisting of:
Ac, FPMP, CEE, TBDMS, TOM, or a photolabile protecting group.
10. The method of claim 9 , wherein PG 1 is a photolabile protecting group.
11. The method of claim 10 , wherein PG 1 is selected from the group consisting of:
NNPOC or MBPMOC.
12. The method of claim 11 , wherein PG 2 is a photolabile protecting group, and wherein the photolabile protecting group of PG 2 is active at wavelengths longer than 365 nm.
13. The method of claim 1 , wherein PG 1 is a photolabile protecting group.
14. The method of claim 12 , wherein PG 1 is selected from the group consisting of:
NNPOC or MBPMOC.
15. A pool of oligonucleotide probes synthesized according to the method of claim 1 , wherein the pool of oligonucleotide probes comprises oligonucleotides of different monomer sequences.
16. The pool of oligonucleotide probes of claim 15 , wherein the pool comprises oligonucleotides of 100 different sequences.
17. The pool of oligonucleotide probes of claim 16 , wherein the pool comprises oligonucleotides of 100,000 different sequences.
18. The pool of oligonucleotides of claim 15 , wherein the different monomer sequences of the oligonucleotide probes are designed for use as polymerase chain reaction primers for a plurality of target sequences.