Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-26
2002-12-03
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S022100, C536S023100, C536S025310, C536S025330, C536S025340
Reexamination Certificate
active
06489466
ABSTRACT:
BACKGROUND
Of all the genes in a genome, only a small fraction are expressed in any individual cell. The temporal and spatial regulation in gene expression determines life processes. Many pathological developments, such as oncogenesis, are driven by gene expression. Identification of multiple genetic alterations as they occur in the genome is critical to understanding the molecular genetic events. Sequence analysis of nucleic acid molecules is essential to understanding and diagnosing disorders. Genetic alterations identified as being related to diseases can then be used as a guide for drug discovery. In the past two decades, the invention of techniques for immobilizing nucleic acid molecules on a solid phase has had a profound impact on the progress of the nucleic acid analysis, such as the use of DNA chips for gene expression analysis and solid phase primer extension for single nucleotide polymorphism analysis. Automation of solid phase oligonucleotide synthesis has helped propel the advancement of molecular biology, which undoubtedly has laid the foundation of the modern understanding of life sciences.
SUMMARY
In general, the present invention features a method for producing an immobilized oligonucleotide attached to a substrate via a C-5′ position and having a terminal C-3′ position.
In one aspect, this invention is directed to a method of producing an immobilized oligonucleotide on a substrate to which a first nucleotide is covalently attached via its C-5′ oxygen. The first nucleotide can be a nucleotide monomer or the 5′ terminal nucleotide of a nucleotide polymer. In general, such a first nucleotide includes a modified nucleotide tethered to a support substrate through a linking group. In particular, the modified nucleotide is constructed such that the C-5′ end of the nucleotide is tetherable to the linking group and the C-3′ end is available for further controlled modification, e.g., addition of other nucleotides in specific sequences to the immobilized nucleotide. In the case of adding a nucleotide monomer as the first nucleotide, the C-3′ end is the C-3′ of the nucleotide monomer. In the case of adding a nucleotide polymer as the first nucleotide, the C-3′ end is the C-3′ of the terminal nucleotide of the polymer. Additionally, the linking group is of sufficient length to allow the immobilized nucleotide to be used to synthesize and screen arrays of nucleotide oligomers, e.g., enzymatic C-3′ primer extension.
In another aspect, the invention provides a method for in situ solid phase oligonucleotide synthesis with C-5′ attached to the substrate, thereby producing oligonucleotides which are a polymer of nucleotides. The method covers an in situ deprotection-activation-coupling cycle of oligonucleotide synthesis that includes covalently coupling a modified nucleotide via its C-5′ oxygen to an immobilized hydroxyl, wherein the modified nucleotide includes a C-3′ photolabile protecting group and a C-5′ hydroxyl group, and also wherein the immobilized hydroxyl group is activated with a phosphorous activating group. The synthesis includes sequentially deprotecting photolabile group from the C-3′ oxygen of an immobilized nucleotide at terminus, activating the C-3′ oxygen at terminus, in situ, with an activating phosphorous group, and coupling C-3′ protected nucleotides to the activated nucleotide at terminus. Optionally, the cycles of deprotecting, activating, and coupling can be repeated until a desired oligonucleotide is obtained. Typically, the immobilized C-3′ oxygen is activated with a phosphorous group such as a phosphoramidite, [(i-Pr)
2
N]POCH
2
CH
2
CN. The produced oligonucleotide can be further involved in enzyme-catalyzed reactions, e.g., polymerase mediated primer extension.
In further another aspect, the invention provides a method for an oligonucleotide synthesis in a direction of 5′ to 3′, thereby producing oligonucleotides that are a set of specific nucleotides. The method covers a deprotection-activation-coupling oligonucleotide synthesis which consists of a nucleotide or an oligonucleotide having a free terminal C-3′ hydroxyl and a terminal C-5′ that is blocked by a group, wherein the free terminal C-3′ hydroxyl is activated with a phosphorous activating group. The synthesis includes sequentially deprotecting a photolabile protecting group from the C-3′ oxygen of a nucleotide at terminus, activating the C-3′ oxygen, in situ, with an activating phosphorous group, and coupling another C-3′ photolabile protected nucleotides to the activated nucleotide at terminus. Optionally, the cycles of deprotecting, activating, and coupling can be repeated until a desired oligonucleotide is obtained. Typically, the C-3′ oxygen is activated with a phosphorous group such as a phosphoramidite, e.g., [(i-Pr)
2
N]POCH
2
CH
2
CN.
The invention also features an array. The array includes a substrate having a plurality of addressable sites. Each of the sites of the plurality has an oligonucleotide covalently attached to the substrate via its C-5′ oxygen atom. Each site of the plurality can be directly adjacent to at least one other site. The sequence of each oligonucleotide of a site can be unique among the plurality. Addressable sites other than the sites of the plurality can be disposed on the array. The array can have a density of addresses and oligonucleotides described below. A spatially selective irradiation technique can be used to make such an oligonucleotide array. Generally, the method covers an in situ deprotection-activation-coupling oligonucleotide synthesis to covalently couple the C-5′ position of a non-immobilized nucleotide or oligonucleotide to a substrate to form an immobilized oligonucleotide having a photolabile group protected C-3′ oxygen available for the attachment of subsequent C-3′ protected nucleotides. The substrate includes a plurality of immobilized nucleotide starters arranged on the substrate as a 2-dimensional array. The synthesis for covalently coupling the C-5′ position of a non-immobilized nucleotide or oligonucleotide to a substrate includes selectively removing photolabile groups from a subset of immobilized nucleotides or oligonucleotides in the array by irradiating the subset to produce an hydroxyl group at the C-3′ terminus. The C-3′ hydroxyl group on the immobilized nucleotide at terminus can be activated again in-situ to form phosphoramidite for coupling the next non-immobilized nucleotide or oligonucleotide having a C-5′ hydroxyl group. Alternatively, the C-3′ hydroxyl group on the immobilized nucleotide can couple with a non-immobilized nucleotide or oligonucleotide having an C-5′ activated group and a C-3′ photolabile protecting group. The sequence and the length of the immobilized oligonucleotide can be chosen for specific applications. Photolabile protecting groups include, but are not limited to, NVOC, MBNPEOC, and MeNPOC.
Within the scope of this invention is a method to synthesize a nucleotide that is activated at C-5′ and photolabile group-protected at C-3′. The method includes protecting the C-5′ hydroxyl group, attaching a photolabile protecting group to the C-3′ oxygen, deprotecting the C-5′ hydroxyl group, and attaching an activated phosphorous group to the C-5′ oxygen of a ribonucleic or deoxyribonucleic acid. Typically, the activated phosphorous group is phosphoramidite, i.e., [(i-Pr)
2
N]POCH
2
CH
2
CN. In general, the stereochemistry at C-1, i.e., where the base is attached to the sugar ring, can be selected to improve the synthetic yield of the C-5′ activated, C-3′ photolabile group protected nucleotide. Also contained within the scope of this invention is a method to covalently couple the C-5′ activated terminus of a monomeric nucleotide to a surface modified functional group on a substrate to form an immob
Huang Tai-Nang
Huang Yih
Shen Ming
Fish & Richardson P.C.
Linden Technologies, Inc.
Riley Jezia
LandOfFree
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