Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-09
2003-04-15
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024500, C536S025300, C536S025330, C536S026600, C435S006120
Reexamination Certificate
active
06548652
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to the chemistry of the attachment of oligonucleotides to solid supports. More particularly the present invention relates to linking oligonucleotides to solid supports through a Schiff base type covalent linkage for capture and detection of single- and double stranded DNA and RNA targets.
BACKGROUND OF THE INVENTION
The detection and quantification of very small quantifies of nucleic acids plays an important role in the biological, forensic and medical sciences. Typically nucleic acids in samples are detected by hybridization to a complementary oligonucleotide containing more than 8 contiguous nucleotides. To provide a signal proportional to the target-oligonucleotide hybrid, typically either the target or the second probe contains a signal generating label, such as a radioactive-, fluorescent-, chemiluminescent-moiety or an enzyme (such as horseradish peroxidase) that through its catalytic activity yields a detectable product. The prior art is well developed in this regard and numerous methods are available for the detection and quantification of signal in the nucleic acid field.
Following the hybridization of the capturing and labeled oligonucleotide to the target nucleic acid it is necessary to separate the signal generating duplex from unreacted target and labeled oligonucleotide. This can usually be accomplished because either the target, or more typically the capturing oligonucleotide has been immobilized on a solid support thereby allowing the isolation of the hybrid free from unhybridized molecules. In a “sandwich assay” variation, an oligonucleotide is immobilized to a solid support and is used to capture a target. The captured target is detected by hybridization with a second labeled oligonucleotide, that has a different sequence than the capturing oligonucleotide.
Numerous types of solid supports suitable for immobilizing oligonucleotides are known in the art. These include nylon, nitrocelluose, activated agarose, diazotized cellulose, latex particles, plastic, polystyrene, glass and polymer coated surfaces. These solid supports are used in many formats such as membranes, microtiter plates, beads, probes, dipsticks etc. A wide variety of chemical procedures are known to covalently link oligonucleotides directly or through a linker to these solid supports. Of particular interest as background to the present invention is the use of glass and nylon surfaces in the preparation of DNA microarrays which have been described in recent years (Ramsay, Nat. Biotechnol., 16: 40-4 (1998)). The journal Nature Genetics has published a special supplement describing the utility and limitations of microarrays (Nat.Genet., 21(1): 1-60 (1999).
Typically the use of any solid support requires the presence of a nucleophilic group to react with an oligonucleotide that must contain a “reactive group” capable of reacting with the nucleophilic group. Alternatively, a “reactive group” is present or is introduced into the solid support to react with a nucleophile present in or attached to the oligonucleotide. Suitable nucleophilic groups or moieties include hydroxyl, sulfhydryl, amino and activated carboxyl groups, while the groups capable of reacting with these and other nucleophiles (reactive groups) include dichlorotriazinyl, alkylepoxy, maleimido, bromoacetyl goups and others. Chemical procedures to introduce the nucleophilic or the reactive groups on to solid support are known in the art, they include procedures to activate nylon (U.S. Pat. No. 5,514,785), glass (Rodgers et al., Anal. Biochem., 23-30 (1999)), agarose (Highsmith et al., J., Biotechniques 12: 418-23 (1992) and polystyrene (Gosh et al., Nuc. Acid Res., 15: 5353-5372 (1987)). Dependent on the presence of either a reactive or nucleophilic groups on the solid support and oligonucleotide, coupling can either be performed directly or with bifunctional reagents. Bifunctional and coupling reagents are well known in the art and many are available from commercial sources.
Of special interest as background to the present invention is the procedure described by Kremsky et al. (Nuc.Acid Res., 15: 2891-2909 (1987)) for the preparation of a 16-mer oligonucleotide containing a 6 carbon carboxylic acid linker on the 5′-end. This product was synthesized using the appropriate phosphoramidites on a standard synthesizer. The acid was then reacted with 3-amino-1,2-propanediol in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to yield a stable diol. The diol was oxidized to the aliphatic aldehyde stage that was subsequently reacted with hydrazide latex beads to form Schiff base linkages that were reduced with sodium cyanoborohydride. The authors indicated that the oligonucleotide diol was a stable intermediate but that the aldehyde should be prepared immediately before coupling to the latex bead to minimize undesirable reaction of the aldehyde with the oligonucleotide bases.
Another article of special interest as background to the present invention is by Tsarev et al. (Biorg.Khim., 16: 765-79 (1990)) that describes coupling of an aromatic aldehyde to the 5′ phosphate of an oligonucleotide through alkylation. The product was used to probe the RNA polymerase promoter complex.
Typically, glass surfaces are activated by the introduction of amino-, sulfhydryl-, carboxyl- or epoxyl-groups to the glass using the appropriate siloxane reagent. Specifically, immobilization of oligonucleotide arrays on glass supports has been described: by Guo et al., Nuc. Acid Res., 22: 5456-5465 (1994) using 1,4-phenylene diisothiocyanate; by Joos et al., Anal. Biochem., 247: 96-101 (1997) using succinic anhydride and carbodiimide coupling; and by Beatti, et al., Mol. Biotech., 4: 213-225 (1995) using 3-glycidoxypropyltrimethoxysilane.
The rapid specific reaction of cytidine in single stranded DNA with semicarbazide moiety containing reagent, in the presence of bisulfite, has also been described (Hayatsu, Biochem., 15: 2677-2682 (1976)).
Procedures which utilize arrays of immobilized oligonucleotides, such as sequencing by hybridization and array-based analysis of gene expression are known in the art. In these procedures, an ordered array of oligonucleotides of different known sequences is used as a platform for hybridization to one or more test polynucleotides, nucleic acids or nucleic acid populations. Determination of the oligonucleotides which are hybridized and alignment of their known sequences allows reconstruction of the sequence of the test polynucleotide. See, for example, U.S. Pat. Nos. 5,492,806; 5,525,464; 5,556,752; PCT Publications WO 92/10588, WO 96/17957 and the scientific publications by Ramsay, Nat. Biotechnol., 16: 40-4 (1998) and by Lipshutz et al., Nat. Genet., 21: 20-24 (1999)).
Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays has been described (Weiler et al, Nucl. Acids Res., 25: 2792-9 (1997). PNAs and PNA/DNA chimeras are also well described.((Nielsen, Curr Opin Biotechnol. 10: 71-5 (1999); Koch et al., Tetrahedron Let., 36: 6933-6936 (1995)).
However, many of the current immobilization methods suffer from one or more of a number of disadvantages. Some of these are, complex and expensive reaction schemes with low oligonucleotide loading yields, reactive unstable intermediates prone to side reactions and unfavorable hybridization kinetics of the immobilized oligonucleotide. The efficient immobilization of oligonucleotides on glass surface in arrays in a high-through put mode requires a) simple reliable reactions giving reproducible loading for different batches, b) stable reaction intermediates, c) arrays with high loading and fast hybridization rates, d) high temperature stability, e) low cost, f) specific attachment at either the 5′- or 3′-end or at an internal nucleotide and g) low background.
The present invention represents a significant step in the direction of meeting or approaching several of these objectives.
SUMMARY OF THE INVENTION
In accordance with the present invention a Schiff base type covalent linka
Hedgpeth Joel
Lukhtanov Eugeny Alexander
Podyminogin Mikhail A.
Epoch Biosciences, Inc.
Riley Jezia
Townsend and Townsend / and Crew LLP
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