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Method of cleaving oligonucleotides off of a microarray while maintaining spatial locations

IP.com Disclosure Number: IPCOM000217084D
Publication Date: 2012-May-01
Document File: 5 page(s) / 28K

Publishing Venue

The IP.com Prior Art Database

Abstract

A library of oligonucleotides can be made by fabricating an array of the oligonucleotides using in situ synthesis methods, and cleaving oligonucleotides from the substrate. In such methods, the nucleotide monomers are sequentially added to a growing nucleotide chain that is attached to a solid support via a cleavable linker. The oligonucleotides can be released from the support by cleaving the cleavable linker. In many cases, thousands of different oligos can be prepared on a glass slide, using a cleavable linker to attach the oligonucleotide to the surface. In many cases, the linker may contain an ester that is cleaved with base (e.g. ammonia, methylamine, ethylenediamine, or ethanolamine) either prior to or simultaneous with removal of the exocyclic amine protecting groups on dA, dG, and dC. Examples of such methods are described in, e.g., Cleary et al (Nature Methods 2004 1: 241-248) and LeProust et al (Nucleic Acids Research 2010 38: 2522-2540).

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Method of cleaving oligonucleotides off of a microarray while maintaining spatial locations

Joel Myerson

Background

A library of oligonucleotides can be made by fabricating an array of the oligonucleotides using in situ synthesis methods, and cleaving oligonucleotides from the substrate. In such methods, the nucleotide monomers are sequentially added to a growing nucleotide chain that is attached to a solid support via a cleavable linker. The oligonucleotides can be released from the support by cleaving the cleavable linker. In many cases, thousands of different oligos can be prepared on a glass slide, using a cleavable linker to attach the oligonucleotide to the surface. In many cases, the linker may contain an ester that is cleaved with base (e.g. ammonia, methylamine, ethylenediamine, or ethanolamine) either prior to or simultaneous with removal of the exocyclic amine protecting groups on dA, dG, and dC. Examples of such methods are described in, e.g., Cleary et al (Nature Methods 2004 1: 241-248) and LeProust et al (Nucleic Acids Research 2010 38: 2522-2540).

These methods, while simple and effective, results in all of the members of the oligonucleotide library being mixed together. This is desirable for applications in which a complex mixture of oligos is the desired end product, as is the case when the entire mixture is to be amplified (with common PCR primers, for example). Assembly of such libraries into larger oligonucleotides (e.g. for gene synthesis) is ultimately limited by the purity of the oligonucleotides and the complexity of the library.

For certain applications it would be desirable to release the oligonucleotides from the surface while keeping their spatial relationship intact (i.e., not have them all mixed together). After release, individual oligonucleotides or groups of oligonucleotides could be manipulated by a variety of techniques, for example by using microfluidics, electrowetting, or direct pipetting for further processing such as PCR or ligation.

For example, for gene synthesis, it is advantageous to perform a convergent synthesis, assembling individual oligonucleotides or sets of oligonucleotides in a particular order, which precludes them being all mixed together. While this could be done by physically dicing a microarray into smaller units followed by conventional cleavage and deprotection for each unit, it would be advantageous to be able to manipulate the oligonucleotides directly on the (undiced) microarray surface.

Description of Method

Oligonucleotide libraries (OLS) can created such that the individual oligonucleotides remain separate after they have been cleaved off the surface and the protecting groups removed. Methods for accomplishing this can be broadly separated into two groups:

1. Those that rely on the creation of physical microwells on the surface in which the cleavage and deprotection chemistry can occur; and

2. Those that use a planar surface, but rely on chemical and physical techniques to keep...