Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-21
2003-01-07
Jones, W. Gary (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S006120, C544S083000
Reexamination Certificate
active
06504019
ABSTRACT:
INTRODUCTION
BACKGROUND
Nucleic acids are set apart from other biomolecules by their ability to hybridize to complementary sequences, a feature that is exploited in nature for the replication of genetic information. Hybridization specificity is exploited in research and diagnostics to generate information about the presence and quantity of nucleic acid sequences. Hybridization assays are generally based on the specific binding of a single stranded analyte to a labeled single stranded probe, followed by detection of the resulting duplexes. Variations of this basic scheme have been developed to enhance specificity, facilitate the separation of the duplexes from extraneous materials, and/or amplify the detectable signal.
The development of solid phase oligonucleotide synthesis has greatly simplified the production of specific nucleic acid probe and primers. Synthetic probes are widely used for all aspects of nucleic acid diagnosis, therapy and investigation. A feature that can be provided only in synthesized probes is comb-type branched multimers, which are composed of a linear backbone and pendant side chains. The backbone includes a segment that provides a specific hybridization site for a nucleic acid of interest, while the pendant side chains include iterations of a segment that provide specific hybridization sites to a second sequence of interest. The branch points are typically provided by protected phosphoramidites, as described in U.S. Pat. No. 5,359,100 (Urdea et al.); U.S. Pat. No. 5,656,731 (Urdea); U.S. Pat. No. 5,124,246 (Urdea et al.) and U.S. Pat. No. 5,710,264 (Urdea et al.), which are introduced during the oligonucleotide synthesis. The branch points may by symmetric or asymmetric.
An appealing aspect of synthetic primers is the ability to tag the probe as it is synthesized, thereby eliminating a separate labeling procedure. Common tags include internal or terminal tags or spacers, where an attached detectable label may be fluorescein or other fluorochromes, or a binding moiety such as biotin, digoxigenin, etc. Spacers known in the art include those with a 2-aminobutyl-1,3-propanediol backbone (U.S. Pat. No. 5,451,463), which is incorporated during phosphoramidite synthesis.
Detection of specific genetic sequences is an area of active research and development. 1X However, many problems still exist, such as low levels of signal, small sample size, high sample complexity, and the like. Improvements in the ability to provide a multiplicity of labels to a specific probe sequence are of interest, particularly using reagents that are compatible with standard phosphoramidite synthesis. The present invention addresses these issues.
RELEVANT LITERATURE
The synthesis of multiple-label carriers using DNA synthesis chemistry is disclosed in U.S. Pat. No. 5,359,100 (Urdea); European Patent EP 0 292 128 (Segev), and WO 90/00622 (Kwiatkowski et al.) The use of triethylene glycol as a building block is described in U.S. Pat. No. 4,914,210 (Leveason et al.) The basic method for solid phase DNA synthesis using phosphoramidite chemistry is described in U.S. Pat. No. 4.458,066, issued Jul. 3, 1984; U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No. 5,153,319, issued Oct. 6, 1992. Reagents and protocols are widely available, for example from Applied Biosystems, Inc. (Foster City, Calif.). Branching phosphoramidites are commercially available, for example from Clontech (Palo Alto, Calif.).
SUMMARY OF THE INVENTION
Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels are provided. The tags are branched structures synthesized using solid phase phosphoramidite chemistry, generally in combination with synthesis of the nucleic acid portion of the probe. The building blocks of the tag are protected glycerol, mono- and di-ethylene glycol phosphoramidites; and reagents that introduce attachment sites for labels. The resulting tag structure permits introduction of multiple labels, while enhancing the hydrophilicity of the probes through additional phosphodiester moieties.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
DEFINITIONS
It is to be understood that this invention is not limited to the particular methodology, protocols, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a probe” includes a plurality of such probes and reference to “the structure” includes reference to one or more such structures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
A probe refers to a biopolymer comprising a nucleic acid moiety and a tag moiety. A labeled probe further comprises one or more detectable label moieties covalently or non-covalently attached to the attachment sites provided by the tag moiety. The nucleic acid sequence is complementary to a nucleic acid sequence of interest present in a target analyte.
As used herein, the term target region or target nucleotide sequence refers to a probe binding region contained within the target molecule. The term target sequence refers to a sequence with which a probe will form a stable hybrid under desired conditions.
The nucleic acid moiety of the probe as used herein is conventional. The length, degeneracy, and specific sequence of the nucleic acid moiety is determined largely by the use for which it is intended. Generally the length of a particular strand will be sufficiently long to provide for specific hybridization of the sequence of interest, and sufficiently short to provide for a difference in hybridization between the sequence of interest and other sequences such as may be present in the sample. For example, detection of a single base change in a genetic sequence may be accomplished with probes of from about 12 to 25 nucleotides in length. Multiple strands may be combined in a comb or fork-like structure.
It will be appreciated that the binding sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form where fewer than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides. Accordingly, as used herein the term “complementary” refers to an oligonucleotide that forms a stable duplex with its “complement” under assay conditions, generally where there is about 90% or greater homology.
The nucleic acid moiety is typically synthesized in vitro using standard chemistry, and may be naturally occurring, e.g. DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoramidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3′—O′—5′—S-phosphorothioate, 3′−S—5′—O-phosphorothioate, 3′—CH
2
—5′—O-phosphonate and 3′—NH—5′—O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.
Sugar modifications are also used to enhance stability and affinity. The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer. The 2′—OH of the ribose sugar may be altered to form 2′—O-methyl or
Ford Donna M.
Law Say-Jong
Monahan John E.
Sells Todd B.
Yang Guohan
Bayer Corporation
Chakrabarti Arun K.
Jones W. Gary
Reed Dianne E.
Reed & Associates
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