Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-04-16
2004-06-01
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S025300, C536S026600
Reexamination Certificate
active
06743905
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates generally to nucleobase polymer functionalizing reagents, to mobility-modified sequence-specific nucleobase polymers, to compositions comprising a plurality of mobility-modified sequence-specific nucleobase polymers, and to the use of such polymers and compositions in a variety of assays, such as, for example, for the detection of a plurality of selected nucleotide sequences within one or more target nucleic acids.
2. BACKGROUND OF THE INVENTION
Methods used to detect selected nucleotide sequences within target nucleic acids underlie an extensive array of practical applications including, but not limited to, paternity testing, forensic analysis, organ donor recipient matching, disease diagnosis, prognosis and treatment, and prenatal counseling.
There exists a need in the art for materials and methods that permit pluralities of selected nucleotide sequences to be simultaneously detected and analyzed, under uniform experimental conditions, preferably in a single, automated, assay reaction. One approach towards meeting this need has been the development of mobility-modifying polymers that can be attached to sequence-specific nucleobase polymers that act to increase the effective size of the modified nucleobase polymers. Where the charge to translational frictional drag ratio of the mobility-modifying polymer differs from that of the nucleobase polymer to which it is attached, the resulting modified nucleobase polymer will have an electrophoretic mobility that differs from that of the unmodified nucleobase polymer. This alteration of the charge to translational frictional drag ratio may be employed in various applications to effect electrophoretic separation of similarly-sized nucleobase polymers under both sieving and non-sieving conditions.
The most commonly employed mobility-modifying polymers are polyethylene oxides (PEO) that are attached to a nucleobase polymer using standard DNA chemistry (Grossman et al. (1994)
Nucleic Acids Research
22 (21): 4527-34). An exemplary standard PEO phosphoramidite reagent (“PEO reagent”) that can be added to a nucleobase polymer using standard DNA chemistry is illustrated below:
In the illustration, DMT represents dimethoxytrityl and iPr represents isopropyl. When x=5, each PEO reagent added to the nucleobase polymer imparts the nucleobase polymer with an electrophoretic retardation of approximately 2 nucleotides as compared to the unmodified nucleobase polymer under both sieving and nonsieving electrophoretic conditions. Due to limitations of DNA chemistry, no more than about 40 PEO reagents can be coupled to a nucleobase polymer and result in homogenous product. Accordingly, the greatest electrophoretic mobility retardation that can be achieved using these standard PEO modifying reagents is about 80 nucleotides.
However, in light of the increasing need to simultaneously analyze vast numbers of nucleotide sequences in a single experiment, e.g., the 200 identified alleles associated with cystic fibrosis, there remains a need in the art for new mobility-modifying polymers that have different charge to translational frictional drag ratios than currently available mobility-modifying polymers, and that can impart electrophoretic mobility retardations of greater than the 80 nucleotides achievable with available PEO modifying reagents. The availability of such new mobility-modifying polymers would greatly increase the repertoire of available mobility modifications, thereby enabling the ability to perform extremely complex sequence analyses in simple, preferably automated, formats.
3. SUMMARY OF THE INVENTION
In one aspect, the present invention provides mobility-modifying phosphoramidite functionalizing reagents comprising a polymeric portion and a phosphoramidite moiety. The phosphoramidite moiety comprises an oxygen-protecting group that, quite unlike the &bgr;-cyanoethyl oxygen protecting group used in conventional phosphoramidite reagents, is stable to basic conditions such as the conditions and reagents used in conventional phosphoramidite oligonucleotide synthesis and deprotection. As a consequence of this stable oxygen protecting group, the bond formed between the functionalizing reagent and the compound functionalized is an uncharged phosphate triester. The mobility-modifying phosphoramidite reagents of the invention may be used to functionalize a wide variety of substances and materials to add mass and size to the substance or material without substantially altering its overall net charge.
The mobility-modifying phosphoramidite reagents of the invention are compatible with standard phosphoramidite synthetic schemes and can be used with commercially available nucleobase polymer synthesis instruments. Thus, the mobility-modifying phosphoramidite reagents of the present invention are particularly convenient for mobility-modifying synthetic sequence-specific nucleobase polymers such as, for example, 2′-deoxyoligonucleotides. They may be readily attached to the 5′-terminus of the nucleobase polymer, to the 3′-terminus, or to both the 5′- and 3′-termini, depending on the particular application and/or desired degree of mobility modification.
The terminus of the polymeric portion that is distal to the phosphoramidite moiety may be protected with a group that is selectively removable under the desired synthesis conditions, or it may comprise a group that is essientially non-reactive, such as, for example an alkyl, aryl, arylakyl, etc. group. In the former embodiment, the protecting group may be selectively removed for sequential condensation of one or more additional phosphoramidite reagents. Suitable selectively removable protecting groups will depend upon the identity of the group being protected and will be apparent to those of skill in the art. Selectively removable groups suitable for protecting hydroxyl groups include, by way of example and not limitation, any of the acid-labile groups that are commonly used to protect the 5′-hydroxyl of conventional nucleoside phosphoramidites oligonucleotide synthesis reagents, such as acid-labile trityl groups (e.g., monomethoxytrityl, dimethoxytrityl, etc.).
The mobility-modifying phosphoramidite reagents of the invention may be used alone to mobility-modify substances such as nucleobase polymers to add mass and size to the nucleobase polymer without altering its overall net charge. Alternatively, they may be used in conjunction with conventional mobility-modifying reagents, such as the PEO reagent illustrated above, to mobility-modify substances such as nucleobase polymers. Because the mobility-modifying phosphoramidite reagents of the invention may be used to add mass and size to a substance such as a nucleobase polymer without altering its overall charge, when used in conjunction with conventional reagents such as PEO reagents, they vastly increase the repertoire of available mobility-modifications that can be added to substances such as nucleobase polymers.
The polymeric portion composing the reagent may be any of a variety of polymers that are soluble under the desired conditions of use and that either include, or can be modified to include, a functional group, such as, for example, a primary hydroxyl group, that can be conveniently converted to a phosphoramidite moiety, typically using standard art-known chemistries. Typical polymers include, but are not limited to, polyoxides, polyamides, polyimines and polysaccharides. The polymers may be used singly or in combinations, such as in the form of copolymers or block polymers. Exemplary polymers include linear or branched polyalkylene oxides, or derivatives thereof, comprising from about 2 to 10 monomer units. Typical derivatives include, for example, those in which the terminal hydroxyl is replaced with a sulfanyl group or an amino group. A useful polyalkylene oxide polymer is polyethylene glycol. The polymer may optionally include a label or other reporter group or molecule, a protecting group for protecting the mobility-modified nucleobase polymer d
Graham Ron
Tian Jing
Woo Sam L.
Applera Corporation
Liptak Vincent P.
Riley Jezia
Ryan Michael J.
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