# Patent

## Title
Oligonucleotide probes and primers comprising universal bases for diagnostic purposes

## Abstract

Aspects of the invention relate to novel oligonucleotides comprising universal and generic bases for use as primers and probes, as well as, methods of using said oligonucleotides for the diagnosis of disease.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/142,729 filed May 8, 2002 now abandoned, which claims priority to U.S. Provisional Application 60/306229, filed Jul. 18, 2001. This application is also a continuation-in-part of application Ser. No. 09/136,080 filed on Aug. 18, 1998 now U.S. Pat. No. 6,518,017, which claims priority to U.S. Provisional Application 60/060,673 filed on Oct. 2, 1997. This application claims priority to all of the aforementioned applications, the disclosures of which are hereby expressly incorporated by reference in their entireties.


FIELD OF THE INVENTION
Aspects of the invention relate to novel oligonucleotides comprising universal and generic bases for use as primers and probes, as well as, methods of using said oligonucleotides for the diagnosis of disease.
BACKGROUND OF THE INVENTION
The explosion of recent knowledge in basic genetics has spawned numerous clinical follow-up studies that have confirmed an unequivocal association between the presence of specific prevalent genetic alterations and susceptibility to some very common human diseases. In addition, the Human Genome Project's sequencing efforts will contribute yet more candidate disease genes that will require both research-based genetic association studies (to confirm suspected disease links) and, if positive, the translation of these disease-genotype associations to routine diagnostic clinical practice. Given this expanding repertoire of confirmed and reputed disease genes (many for common diseases), the demand for rapid, sensitive, specific, inexpensive assays for their clinical- and/or research-based detection is growing quickly.
As a consequence, clinical genetic testing laboratories, once accustomed to manual, low-volume, high-labor tests on patients with rare, untreatable classic “genetic” diseases, will soon need to develop better high-throughput and semi-automated methods. In the fast-approaching molecular medicine era, these new genotyping methods will be utilized not only for diagnosing symptomatic patients but perhaps, more importantly, for presymptomatically identifying individuals at risk for common, treatable diseases for whom effective preventative interventions may be available.
Oligonucleotide hybridization is a method commonly used in the field of molecular biology for the treatment and diagnosis of disease, as well as the identification, quantitation, and isolation of nucleic acids. Accordingly, it is important to identify methods to increase the specificity and affinity of oligonucleotides for their targets. In this way, diagnostics which provide efficient and precise answers can be made. Various methods for increasing the specificity of oligonucleotides are known in the art, including increasing the length, choosing oligonucleotides that are not likely to cross-hybridize or bind non-specifically and designing oligonucleotides that have a high annealing temperature. (See e.g., Bergstrom et al., J. Am. Chem. Soc. 117:1201-1209, 1995; Nicols et al., Nature 369:4920493, 1994; Loakes, Nucl. Acids Res. 22:4039-4043, 1994; Brown, Nucl. Acids Res. 20:5149-5152, 1992).
Recently, investigators have determined that modified oligonucleotides containing universal bases provide some benefit over conventional oligonucleotide chemistries. (See Guo et al., U.S. Pat. No. 5,780,233, filed Jun. 6, 1996). Although Guo et al., observed some improvement in being able to discriminate a variant nucleotide in a target nucleic acid by incorporating solitary universal bases (artificial mismatches) sprinkled throughout a probe oligonucleotide, particular spacing and composition requirements were necessary. For example, Guo et al. found that the universal base should be carefully spaced from the variant nucleotide (i.e., 3 or 4 nucleotides away) and that the oligonucleotide probes should not contain a total composition of universal bases of greater than 15%.
Van Ness et al. (U.S. Pat. No. 6,361,940, filed Apr. 1, 1998) also found that the incorporation of universal bases (specificity spacers) could increase the specificity of a probe oligonucleotide for a target nucleic acid. As above, however, Van Ness et al. determined that the universal bases should be spaced a considerable distance from each other (4-14 nucleotides). Thus, despite the advances made by the investigators above and others in the field, there still remains a need for better oligonucleotide chemistries, which allow for the development of more efficient diagnostics and therapeutics.
SUMMARY OF THE INVENTION
Aspects of the invention concern oligonucleotides having universal or generic bases, which can be used for diagnostic and therapeutic purposes. Unexpectedly, it was discovered that oligonucleotides having a universal or generic base composition of at least 20%-50% of the total number of bases facilitate the identification of mutations and polymorphisms, in particular single nucleotide polymorphisms (SNps). Further, it was also discovered that oligonucleotides having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more juxtaposed universal or generic bases facilitate the identification of mutations and polymorphisms, in particular SNPs. The oligonucleotides described herein have many other utilities besides the detection of SNPs including, but not limited to, application in other diagnostic processes, array technology, sequencing, hybridization and other techniques, which use conventional oligonucleotides.
It is further contemplated that placing an unnatural base that has a modified affinity, preferably a higher affinity, but a lower affinity may also be used, increases the ability to differentiate a single nucleotide polymorphism or a polymorphic site from a normal site.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the detection of a single nucleotide base change by quantification of melting temperatures.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Embodiments of the invention concern oligonucleotides that contain universal or generic bases and/or other unnatural bases and methods of using such oligonucleotides to diagnose and treat various diseases.
In some contexts, the term “universal base” is used to describe a moiety that may be substituted for any nucleic acid base. The universal base need not contribute to hybridization, but should not significantly detract from hybridization, whereas “generic bases” are bases that are capable of binding to more than one type of nucleotide. For example a base might be generic for the purine bases or alternatively a base might be generic for the pyrimidine bases. Preferred universal or generic bases include 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, 2-deoxynebularine, dP, or dK derivatives of natural nucleotides. Some embodiments may also utilize degenerate bases. The term “degenerate base” refers to a moiety that is capable of base-pairing with either any purine, or any pyrimidine, but not both purines and pyrimidines. Exemplary degenerate bases include, but are not limited to, 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (“P”, a pyrimidine mimic) and 2-amino-6-methoxyaminopurine (“K”, a purine mimic). In some aspects of the invention, these universal, generic, or degenerate bases are juxtaposed in blocks of artificial bases and in others, they are clustered at either the 5′ or 3′ end of the oligonucleotide or both. Desirably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 universal, generic, or degenerate bases are juxtaposed in each block and an oligonucleotide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 blocks depending on the length of the oligonucleotide and the desired effect. Further, some embodiments contain a non-nucleic acid linker such as a spacer 9, spacer 18, spacer C3, or an abasic spacer such as dSpacer so as to provide greater flexibility in the molecule. In some contexts, these spacers are also referred to as universal bases. Preferably, the oligonucleotides described herein are used to increase the efficiency and specificity of existing diagnostic and therapeutic approaches.
It is further contemplated that substituting an unnatural base for a natural base within the oligonucleotide that has a modified affinity, preferably a higher affinity, but a lower affinity may also be used, increases the ability to differentiate a single nucleotide polymorphism or a polymorphic site from a normal site.
As shown herein, the incorporation of universal or generic bases in an oligonucleotide facilitates the differentiation of nucleic acids that differ by as little as a single nucleotide. In fact, it was discovered that the presence of five universal bases decreased the melting temperature of probe-template hybrids by 17° C., as compared to a 6° C. difference when using conventional oligonucleotides. The oligonucleotides of the invention were found to be very specific and exhibit a high affinity for target. By “target” is meant a natural nucleic acid to be detected, quantified, or amplified, etc., consisting of either DNA or RNA, amplified or unamplified and single-stranded or duplex.
Embodiments of the invention include oligonucleotides having at least 20% universal, generic or a mixture of universal and generic bases. Other embodiments include oligonucleotides having at least 21%, 22%, 25%, 30% or 50% universal, generic or a mixture of universal and generic bases. Still more embodiments are oligonucleotides with at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, or more universal, generic or a mixture of universal and generic bases and unnatural bases located at the SNP position to enhance discrimination. Preferred universal or generic bases are 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, 2-deoxynebularine, dP, or dK derivatives of natural nucleotides. Some embodiments may also utilize degenerate bases. The term “degenerate base” refers to a moiety that is capable of base-pairing with either any purine, or any pyrimidine, but not both purines and pyrimidines. Exemplary degenerate bases include, but are not limited to, 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (“P”, a pyrimidine mimic) and 2-amino-6-methoxyaminopurine (“K”, a purine mimic). In some aspects of the invention, these universal, generic, or degenerate bases are juxtaposed and in others, they are clustered at either the 5′ or 3′ end of the oligonucleotide or both. Desirably, at least three, four, five, six, seven, or eight universal, generic, or degenerate bases are juxtaposed. Further, some embodiments contain a non-nucleic acid linker such as a spacer 9, spacer 18, spacer C3, or an abasic spacer such as dSpacer and with or without unnatural SNP maximum discrimination base.
The oligonucleotides described herein may also contain natural bases or unnatural base analogs that hydrogen bond to natural bases in the target nucleic acid. Additionally, the oligonucleotides described herein may contain natural bases or unnatural base analogs or other modifications that have a lower affinity to or ability to hydrogen bond to natural bases, relative to any natural base. By “non-naturally occurring base” is meant a base other than A, C, G, T and U, and includes degenerate and universal bases as well as moieties capable of binding specifically to a natural base or to a non-naturally occurring base. Non-naturally occurring bases include, but are not limited to, propynylcytosine, propynyluridine, diaminopurine, 5-methylcytosine, 7-deazaadenosine and 7-deazaguanine. In still more embodiments, the oligonucleotides described above have at least two high affinity domains and one or more low affinity domains.
Embodiments of the invention include oligonucleotides having universal, generic or a mixture of universal and generic bases which are juxtaposed. Preferably, the number of juxtaposed bases is 2 or more. In one embodiment, the number of juxtaposed bases is 4 or more, including but not limited to, 5 or more, 6 or more, 7 or more, and 8 or more. The juxtaposed bases may substitute for any natural base and may substitute for a variety of different natural bases. The juxtaposed bases may be as close as 1 nucleotide from a mismatch or may include the mismatch. Another embodiment concerns a method of increasing the specificity of an oligonucleotide by substituting at least 4 juxtaposed nucleic acids with universal or generic bases. Another embodiment concerns a method of increasing the specificity of an oligonucleotide by substituting at least 5, 6, 7 or more juxtaposed nucleic acids with universal or generic bases.
Embodiments of the invention also include methods of making and using the oligonucleotides described above. For example, one embodiment concerns a method of designing an oligonucleotide, which involves identifying a sequence that corresponds to or complements a target sequence and substituting four or more bases within said sequence with universal or generic bases and with or without unnatural SNP maximum discrimination bases. Another embodiment concerns a method of increasing the specificity of an oligonucleotide by substituting at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or 40% of the total number of bases with universal or generic bases. A further embodiment concerns a method of increasing the specificity of an oligonucleotide by substituting at least 35%, 40%, 45%, 50%, 55%, 60%, or 70% of the total number of bases with universal or generic bases.
The oligonucleotides described herein, though clearly useful for the identification of single nucleotide polymorphisms (SNP's), are also useful for other conventional methods that employ oligonucleotides (e.g., diagnostics, hybridization, sequencing, etc). The oligonucleotides described herein can be used in most methods known to one of skill in the art in which conventional oligonucleotides are used. Although preferred methods concern the use of said oligonucleotides to detect SNPs, embodiments of the invention also encompass the use of said oligonucleotides as primers (e.g., in conjunction with the TaqmanTM assay, PCR, or RT-PCR), as probes (e.g., in conjunction with the HPSATM, Molecular BeaconTM, HybProbeTM, CPTTM and InvaderTM assays, northern, Southern, or library hybridizations), in arrays (e.g., chip-based arrays, peptide/nucleic acid virtual arrays, DNA microarrays, antisense scanning arrays, or plate-type arrays) and in other techniques involving oligonucleotides (e.g., 5′ or 3′ RACE or related techniques). The term “probe” is used herein to mean an oligonucleotide to detect a target nucleic acid, whereas, the term “primer” is used to refer to an oligonucleotide, which can be used to amplify or extend a target nucleic acid. Thus, several embodiments concern diagnostic methods that employ the embodied oligonucleotides in conjunction with a conventional diagnostic technique.
By one approach, a method of detecting the presence or absence of a mutation or polymorphism in a sample comprising nucleic acids is practiced by contacting said nucleic acid with at least one of the oligonucleotides described above, and identifying whether said oligonucleotide binds to said nucleic acid. Preferably, the universal or generic bases of said oligonucleotides are not located at the site or sites of mutation or polymorphism but unnatural bases allowing higher SNP discrimination might be. Additionally, this method can incorporate an amplifying step (e.g., PCR or RT-PCR) to aid in the identification of the presence or absence of the mutation or polymorphism. The section below describes the oligonucleotides of the invention in greater detail.
Oligonucleotides
The oligonucleotides can be of virtually any sequence and of any length, wherein said oligonucleotides comprise at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more or up to and including 50% universal or generic bases. The term “oligonucleotide” is used to refer to a molecule consisting of DNA, RNA, or DNA/RNA hybrids with or without non-nucleic acid analogues and polymers. In some embodiments the universal or generic bases are juxtaposed and, in others, clusters of at least two universal or generic bases are sprinkled throughout the oligonucleotide sequence. Preferred sequences correspond to already existing probes, which can be used to identify the presence or absence of a SNP or other genetic marker that has an association with a disease. Preferred sequences, for example, include sequences that indicate a predilection to contract cystic fibrosis (See e.g., U.S. Pat. No. 6,201,107, hereby expressly incorporated by reference in its entirety), sickle cell anemia (See e.g., U.S. Pat. No. 4,683,194, hereby expressly incorporated by reference in its entirety), hemochromatosis (See e.g., U.S. Pat. No. 6,025,130, hereby expressly incorporated by reference in its entirety), and cancer (See e.g., U.S. Pat. No. 6,194,158, hereby expressly incorporated by reference in its entirety). It should be understood that other sequences known by those of skill in the art, which indicate a predilection to disease can be used to generate the oligonucleotides of the invention.
Oligonucleotide synthesis is well known in the art, as is synthesis of oligonucleotides containing modified bases and backbone linkages. In fact, such oligonucleotides can often be obtained from commercial suppliers upon providing the supplier with the specific sequence and composition information and a request for custom production. Although the preferred length of the oligonucleotides is less than 100 bases, embodiments can be from about 5 to about 500 nucleotides in length, desirably, 10 to about 300 nucleotides in length, more desirably 12 to about 200 nucleotides in length, preferably, 15 to about 100 nucleotides, more preferably 17 to about 50 nucleotides, and most preferably, about 20 to about 40 nucleotides in length.
The oligonucleotides can employ any backbone and any sequence capable of resulting in a molecule that hybridizes to target DNA and/or RNA. Examples of suitable backbones include, but are not limited to, phosphodiesters and deoxyphodiesters, phosphorothioates and deoxyphosphorothioates, 2′-O-substituted phosphodiesters and deoxy analogs, 2′-O-substituted phosphorothioates and deoxy analogs, morpholino, PNA (U.S. Pat. No. 5,539,082, hereby expressly incorporated by reference in its entirety), 2′-O-alkyl methylphosphonates, 3′-amidates, MMI, alkyl ethers (U.S. Pat. No. 5,223,618, hereby expressly incorporated by reference in its entirety) and others as described in U.S. Pat. Nos. 5,378,825, 5,489,677 and 5,541,307, all of which are hereby expressly incorporated by reference in its entirety. Where RNase activity is desired, a backbone capable of serving as an RNase substrate is employed for at least a portion of the oligonucleotide.
Universal or generic bases suitable for use with the embodiments described herein include, but are not limited to, deoxy 5-nitroindole, deoxy 3-nitropyrrole, deoxy 4-nitrobenzimidazole, deoxy nebularine, deoxyinosine, 2′-Ome inosine, 2′-Ome 5-nitorindole, 2′-Ome 3-nitropyrrole, 2′-F inosine, 2′-F nebularine, 2′-F 5-nitroindole, 2′-F 4-nitrobenzimidazole, 2′-F 3-nitropyrrole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2′-O-methoxyethyl inosine, 2′O-methoxyethyl nebularine, 2′-O-methoxyethyl 5-nitroindole, 2′-O-methoxyethyl 4-nitro-benzimidazole, 2′-O-methoxyethyl 3-nitropyrrole, deoxy RpMP-5-nitroindole dimer 2′-Ome RpMP-5-nitroindole dimer and the like.
Many of the embodied oligonucleotides are characterized in that they share the formula: “XRY”, wherein “X” consists of about 5-10, 11-20, or 5-20 modified nucleic acid bases; “R” consists of about 3-5, 6-10, or 3-10 juxtaposed universal or generic bases; and “Y” consists of about 3-5, 6-10, 11-15, or 3-20 nucleic acid bases; wherein X, R, and Y are covalently joined and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases and X and/or Y might contain a natural or unnatural base at the SNP sight and X and/or Y might contain higher or lower affinity bases or analogues.
Other embodiments include oligonucleotides with the formula: “XRY”, wherein “X” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 modified nucleic acid bases or base analogs that have a lower affinity than natural bases; “R” consists of about 3-5, 6-10, 11-15, 16-20, or 3-20 juxtaposed universal or generic bases; and “Y” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; wherein X, R, and Y are covalently joined and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases.
Still other embodied oligonucleotides have the formula: “XRZRY”, wherein “X” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; “R” consists of about 3-5, 6-10, 11-15, 16-20, or 3-20 juxtaposed universal or generic bases; “Z” consists of about 5-10, 11-20, or 5-20 modified nucleic acid bases; and “Y” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; wherein X, R, Z, and Y are covalently joined and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases.
Still other embodied oligonucleotides have the formula: “XZRZY”, wherein “X” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; “R” consists of about 3-5, 6-10, 11-15, 16-20, or 3-20 juxtaposed universal or generic bases; “Z” consists of about 5-10, 11-20, or 5-20 modified nucleic acid bases, which have a lower or higher affinity than natural bases; and “Y” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; wherein X, R, Z, and Y are covalently joined and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50%, of the total number of bases are universal or generic bases.
More embodied oligonucleotides have the formula: “XZXRXZX”, wherein “X” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; “R” consists of about 3-5, 6-10, 11-15, 16-20, or 3-20 juxtaposed universal or generic bases; “Z” consists of about 5-10, 11-20, or 5-20 modified nucleic acid bases, which have a lower or higher affinity compared to natural bases; wherein X, R, and Z are covalently joined and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases.
Still more embodied oligonucleotides have the formula: “YXZXRY”, wherein “X” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; “R” consists of about 3-5, 6-10, 11-15, 16-20, or 3-20 juxtaposed universal or generic bases; “Z” consists of about 5-10, 11-20, or 5-20 modified nucleic acid bases, which have a lower or higher affinity than natural bases; and “Y” consists of about 5-10, 11-20, 21-30, 31-40, 41-50, or 5-50 nucleic acid bases; wherein X, R, Z, and Y are covalently joined, at least two nucleotides of Y are covalently linked by a non-nucleic acid linker, and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases.
The oligonucleotides described herein can be sold separately or can be incorporated in kits that facilitate genetic analysis. For example, many SNP diagnostic kits are currently available. These kits typically provide oligonucleotide primers, which are to be used to detect a specific SNP that is associated with a disease. Aspects of the invention include diagnostic kits comprising probes and primers that are manufactured in accordance with the oligonucleotide structures described herein. That is, embodiments of the invention include diagnostic kits comprising an oligonucleotide, wherein at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases of said oligonucleotide are universal or generic bases and may or may not contain other unnatural bases. The kits may optionally provide a support (e.g., nitrocellulose, nylon, plastic, or other macromolecule) hybridization or amplification reagents, and instructions. The section below describes in greater detail many of the methods concerning the oligonucleotides described herein.
Methods
Embodiments of the invention also include methods of making and using the oligonucleotides described above. One embodiment concerns a method of designing an oligonucleotide, which involves identifying a sequence that corresponds to or complements a target sequence and substituting sufficient bases within said sequence with universal or generic bases so as to achieve an overall composition in which at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or up to and including 50% of the total number of bases are universal or generic bases. By one approach, a sequence that interacts with a target that indicates the presence or absence of a disease is selected from U.S. Pat. No. 6,201,107; 4,683,194; 6,025,130; or 6,194,158 (all of which are hereby expressly incorporated by reference in their entireties) and at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the total number of bases are swapped with universal or generic bases. Preferably, all of the universal bases are juxtaposed or are clustered at either the 5′ or 3′ end of the oligonucleotide. Care should be taken such that the diagnostic site (e.g., site of the SNP or mutation) is not covered by the universal bases, but may be covered by an unnatural base to enhance SNP discrimination. That is, preferably, the diagnostic site is complemented by a natural base and the stretch of universal or generic bases are positioned such that an optimal difference in melting behavior between the polymorphic/probe complex and non-polymorphic target/probe complex is achieved.
The oligonucleotides described herein, though clearly useful for the identification of single nucleotide polymorphisms (SNP's), are also useful for other conventional methods that employ oligonucleotides (e.g., diagnostics, hybridization, sequencing, etc). The oligonucleotides described herein can be used in most methods known to one of skill in the art in which conventional oligonucleotides are used.
Thus, the oligonucleotides described herein are useful for the identification of any mutations, allelic variants, polymorphisms, and the normal or wild-type sequence of a gene. In addition, the oligonucleotides described herein may be used to detect the presence of a sequence, or alternatively, the oligonucleotides may be used to identify the amount of a particular mRNA with is being produced by a cell. The quantitation may be in addition to, or separately from the identification of the presence.
However, because the most common type of human genetic variation is the single-nucleotide polymorphism (SNP), a base position at which two alternative bases occur at appreciable frequency (>1%) in the population, the utilization of SNPs for clinical diagnostics, whole-genome linkage disequilibrium screens, determination of the recent evolutionary history of a species, and the process of speciation has become a major focus of human genetics. Thus, methods of genotyping or determining the presence or absence of a mutation or polymorphism, preferably a SNP, using the oligonucleotides described herein are extremely useful embodiments.
A prototypical example of the forthcoming primary public health role of molecular diagnostics (particularly of SNPs) is the identification of individuals affected by or at-risk for the iron overload disorder hereditary hemochromatosis. More than 90% of the cases of this most common of all single-gene disorders (present in 0.5% of whites) are caused by the presence of a homozygous well-conserved single nucleotide substitution (nucleotide G845A; amino acid C282Y) in the transferrin receptor binding protein HFE. This loss-of-function mutation abolishes HFE's usual cell surface expression, thus preventing its ability to down-regulate the affinity of transferrin receptor for transferrin-bound iron.
The result is a dysregulation of normal cellular iron metabolism and a resulting constitutive intestinal iron absorption. This excess toxic iron deposits in numerous organs and, if not removed, causes progressive chronic damage to the liver, heart, endocrine glands, joints, and skin. Because hemochromatosis is a common, underdiagnosed (but easily diagnosable), progressive chronic disease with late-onset symptomotology for which an effective, safe (preventative) therapy is widely available (phlebotomy), it is perhaps the ideal disease for the implementation of a population-based screening program.
Universal population-based hemochromatosis screening by transferrin saturation has been recommended by the College of American Pathologists, and a more conservative phenotypic screening of “symptomatic” individuals has been recommended by an expert consensus panel of the Centers for Disease Control (CDC) and the National Institutes of Health (NIH). More universal recommendations for widespread population screens may result from the recently initiated NIH study (HEIRS) of 100 000 apparently healthy Americans that will evaluate the benefits and risks of iron overload screening by both genotypic and phenotypic determinations. Therefore, the need to identify this SNP and diagnose this debilitating disease early on is manifest.
Accordingly, an individual at risk for hemochromatosis can be identified by selecting probes or primers that allow for the detection of the well-conserved single nucleotide substitution, nucleotide G845A. (See e.g., U.S. Pat. No. 6,025,130, hereby expressly incorporated by reference in its entirety, wherein specific primers and probes can be obtained). Once suitable primers are selected they can be designed to have a sufficient amount of universal or generic bases to allow for optimal melting behavior discrimination. The oligonucleotides having universal or generic bases can then be used to identify whether said individual has the mutation that indicates the disease.
In a similar fashion, an individual at risk for cystic fibrosis (CF) can be identified (suitable primers or probes are identified in U.S. Pat. No. 6,201,107), an individual at risk of contracting cancer can be identified (suitable primers and probes are identified in U.S. Pat. No. 6,194,158, hereby expressly incorporated by reference in its entirety), and an individual at risk for sickle cell anemia can be identified (suitable primers and probes are identified in U.S. Pat. No. 4,683,194, hereby expressly incorporated by reference in its entirety).
Although some preferred methods concern the use of said oligonucleotides to detect SNPs, the embodied oligonucleotides can also be used as primers (e.g., in conjunction with the TaqmanTM assay, PCR, or RT-PCR), as probes (e.g., in conjunction with the HybProbeTM, CPTTM and InvaderTM assays, northern, Southern, or library hybridizations), in arrays (e.g., chip-based arrays, peptide/nucleic acid virtual arrays, DNA microarrays, antisense scanning arrays, or plate-type arrays) and in other techniques involving oligonucleotides (e.g., 5′ or 3′ RACE or related techniques).
The following examples describe in greater detail techniques that can be used to make the oligonucleotides described herein.
EXAMPLE 1
By one approach, the oligonucleotides described herein were made using a Perkin-Elmer Applied Biosystems Expedite synthesizer. All reagents were used dry (<30 ppm water) and the oligonucleotide synthesis reagents were purchased from Glen Research. Amidites of normal bases, or universal bases, in solution were dried over Trap-paks (Perkin-Elmer Applied Biosystems, Norwalk, Conn.). A solid support previously derivatized with a dimethoxy trityl (DMT) group protected propyl linker was placed in a DNA synthesizer column compatible with a Perkin-Elmer Applied Biosystems Expedite synthesizer (1 mmol of starting propyl linker). The DMT group was removed with a deblock reagent (2.5% dichloroacetic acid in dichloromethane). The standard protocols for RNA and DNA synthesis were applied to the amidites (0.1 M in dry acetonitrile). The amidites were activated with tetrazole (0.45 M in dry acetonitrile). Coupling times were typically up to 15 minutes depending on the amidite. The phosphonite intermediate was treated with an oxidizing Beaucage sulfurizing reagent.
After each oxidation step, a capping step was performed, which places an acetyl group on any remaining uncoupled 5′-OH groups by treatment with a mixture of two capping reagents: CAP A (acetic anhydride) and CAP B (n-methylimidazole in THF). The cycle was repeated a sufficient number of times with various amidites to obtain the desired oligonucleotide sequence.
Once the desired sequence was obtained, the support was treated at 55° C. in concentrated ammonium hydroxide for 16 hours. The solution was concentrated on a speed vac and the residue was taken up in 100 ml aqueous 0.1 ml triethylammonium acetate. This material was then applied to an HPLC column (C-18, Kromasil, 5 mm, 4.3 mm diameter, 250 mm length) and eluted with an acetonitrile gradient (solvent A, 0.1 M TEAA; solvent B, 0.1 M TEAA and 50% acetonitrile) over 30 minutes at 1 ml/min flow rat. Fractions containing greater than 80% pure product are pooled and concentrated. The resulting residue was taken up in 80% acetic acid in water to remove the trityl group and reapplied to a reverse phase column and purified as described above. Fractions containing greater than 90% purity were pooled and concentrated. By following the approach described above with modifications that are apparent to one of skill in the art, the oligonucleotides described herein can be made, isolated, and purified. The following example describes several preferred structures for designing the embodied oligonucleotides.
EXAMPLE 2
Several motifs that facilitate the identification of SNPs were discovered and this example describes these structures in greater detail. The oligonucleotide motifs are described using the following letter identifications:
N=Natural bases or unnatural base analogues in the oligonucleotide that hydrogen bond to natural bases in the target nucleic acid. N may be higher or lower affinity than natural bases due to base, sugar, backbone, or any other non-nucleic acid modifications or structures, (e.g. peptide nucleic acids).
S=Natural bases or unnatural base analogs or other modification that has a lower affinity to or ability to hydrogen bond to natural bases, relative to any natural base. These bases can stack in the duplex, but have lower affinity to specific opposing natural bases.
B=Any “Universal” or “generic” base analogues or other modification that can stack in duplex nucleic acid helices but do not significantly discriminate among opposing natural bases (universal, e.g. 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, 2-deoxynebularine) or that have a reduced ability to discriminate among opposing natural bases (generic, e.g. dP or dK).
X=Natural base or unnatural base substitution or any other modification within the oligonucleotide that increases the negative impact of a mismatch against the target nucleic acid. X can occur in any region of the oligonucleotide.
L=Non-nucleic acid linker (e.g. Spacer 9, Spacer 18, Spacer C3, dSpacer, all from Glen Research) either as a base substitution or contained between any pair of bases in the probe.
Representative classes of oligonucleotides for use with many of the embodiments described herein are shown below.











(  1  )(  2 )(  3  )



1.
NNNNNNNBBBBBBNNNNNNN


 




(  1  )(  2 )(  3  )



2.
 NNNLNNNBBBBBBNNNNNN


 




(  1  )(  2 )(  3  )



3.
 NNNNNNNLBBBBBNNNNNNN


 




(  1  )(  2 )(  3  )



4.
 NNNNNNLBBBBBBLNNNNNN


 




(  1  )(  2 )(  3  )



5.
 NNNNNNNBBBBBBNNNLNN


 




( 1 )(2)(3)(4)( 5 )



6.
 NNNNNBBBNNNBBBNNNNN






TABLE 1 describes the unnatural and natural base choices that allow one to 1) discriminate SNP bases more precisely that natural bases alone, and 2) create the higher and lower affinity blocks included in the oligonucleotides of the preferred embodiment.








TABLE 1







Natural
NATURAL BASE TO AVOID BINDING











Base
G
A
T
C





G
—
*N4-Et-dC
dC
dC



—
##not 5-Me-dC
5-Me-dC
5-Me-dC



—
##not dC




A
2-Thio-dT
—
2-Thio-
2-Thio-dT



not dT
—
dT





—




T
**2-amino-dA
2-amino-dA
—
2-amino-P



not dA

—
#not 2-





—
amino-dA






#not dA


C
dG
***dX
dX
—




not dG
not dG
—






—





Relative binding strength estimates contributing to choices:


*5-Me-dC: dG > dC: dG > N4-Et-dC: dG? > ?N4-Et-dC: dA


##dT: dA = 5MedC: dA > dC: dA = dU: dA


**2-amino-dA: dT > dA: dT >> 2-amino-dA: dG


***dX: dC = dA: dU >> dX: dG < dA: dG = dA: dI


#2-amino-dA: dC > dA: dC = dA: dU >> dA: dI






It is further contemplated that placing an unnatural base that has a modified affinity, preferably a higher affinity, but a lower affinity may also be used, increases the ability to differentiate a single nucleotide polymorphism or a polymorphic site from a normal site.
Table 1 shown above is designed to exemplify the way any natural or unnatural base or analogue can be selected to maximize SNP discrimination in combination with universal or generic bases. Given any of the general structure permutations shown above (numbered 1-6), for any SNP in any position, Table 1 allows one to determine which base to use for the specific SNP base. For example, it can be used to determine which base one wants this probe to bind to in the target versus the SNP base in the non-target. Most wild-type versus mutant SNP detection systems have both wild-type and mutant targets in the mixture, so one has to absolutely maximize the ability to discriminate the two SNP bases that define wild-type versus mutant and the Table allows one to do so. If one were trying to get better discrimination between an adenine in the wt target and guanine in the mutant target (the SNP), one could go to the table and look up “adenine” as the natural base and under the heading “guanine”, one finds “2-Thio-dT” which tells you that you will get the best discrimination between “A” and “G” if “2-Thio-dT” is used in the primer. This is further illustrated in Examples 12 and 13.
The next example illustrates that the incorporation of universal or generic bases in an oligonucleotide facilitates the differentiation of two sequences that differ by a single nucleotide.
EXAMPLE 3
In these experiments it was demonstrated that oligonucleotides having universal bases facilitate the identification of a single nucleotide base change in a nucleic acid. In a first set of experiments, the differences in melting behaviors of a natural probe/target complex and an oligonucleotide probe having 5 universal bases/target complex was ascertained. The mutant target contained a single mismatch, a G - - G mismatch to both probes, OGC2 and OGX2.
As shown in FIG. 1, the all-natural probe OGC2 (SEQ ID NO: 2) bound to the mismatch target #1090 (SEQ ID NO: 8) with a differential melting temperature of −6° C. relative to the perfect match wild-type target #1088 (SEQ ID NO: 7). OGX2 (SEQ ID NO: 4), the oligonucleotide containing 5 universal bases, bound with a differential melting temperature of −17° C. relative to the perfect match. The slight difference of −6° C. in melting temperature using the control probe (OGC2) is undetectable using most diagnostic methods. In the presence of five universal bases, however, the single purine-purine mismatch decreases the perfect-probe-to-target melting temperature by 17° C., thereby facilitating the specific detection of the SNP in the target oligonucleotide.
Multiple melting temperature determinations were performed for each probe/target combination. All mixtures were heated to 85-95° C. for 10-15 minutes and allowed to cool to room temperature before use. Melting temperatures were determined by UV absorbence in sealed quartz cuvettes using a Varian Cary 3E UV-Visible Spectrophotometer with a Varian Cary temperature controller, controlled with Cary 01.01(4) Thermal software. Temperature gradients decreased from 85° C. to 25° C. at 1° C. per minute. The following example details experiments that examined the effect of salt concentration on the oligonucleotides described herein.
EXAMPLE 4
Melting temperatures were determined for the following three probes containing generic and universal bases in various salt concentrations and the results were compared to those obtained using a control probe without the generic and universal bases (5′ natural OGC2). The probes analyzed included 5′ OGX1 (SEQ ID NO: 3), 5′OGX3 (SEQ ID NO: 5), 5′ OGX5 (SEQ ID NO: 6) and 5′ natural OGC2 (SEQ ID NO: 2). The target was #1088 (SEQ ID NO: 7). Oligonucleotide probes and DNA targets were at 0.35 to 0.40 O.D. each per milliliter in both an enzymatically relevant buffer system (KCl/Mg++) or in a non-physiological, high salt buffer system (NaCl/PO4−−):













KCl/Mg++ Buffer:
NaCl/PO4−− Buffer:








20 mM Tris-HCl, pH = 7.5 at 20° C.
10 mM NaH2PO4,




pH = 7.0 at 20° C.



100 mM KCl
1 M NaCl



10 mM MgCl2
0.1 EDTA



0.05 mM DTT




2.5% w/v sucrose









Multiple melting temperature determinations were performed for each probe/target combination. All mixtures were heated to 85-95° C. for 10-15 minutes and allowed to cool to room temperature before use. Melting temperatures were determined by UV absorbence in sealed quartz cuvettes using a Varian Cary 3E UV-Visible Spectrophotometer with a Varian Cary temperature controller, controlled with Cary 01.01(4) Thermal software. Temperature gradients decreased from 85° C. to 25° C. at 1° C. per minute.
As shown in TABLE 2, the difference in melting behavior of oligonucleotides having universal or generic bases and natural oligonucleotides were not influenced by salt concentration.









TABLE 2








KCl/Mg++
NaCl/PO4−−












Match
MisMatch
Match
MisMatch


Probe
TM
TM
TM
TM





5′ OGX1
<25
53
<25
58


5′ OGX3
<25
51
<25
57


5′ OGX5
<25
56
<25
63


5′ natural OGC2
  64
70
  71
75









The following example demonstrates that the oligonucleotides described herein can be used to detect single base changes in polyacrylamide gel electrophoresis detection systems.
EXAMPLE 5
The melting behavior of control probes (i.e., no universal and generic bases) OGC1 (SEQ ID NO: 1) and OGC2 (SEQ ID NO: 2) annealed to two different target DNA's: #1088 (SEQ ID NO: 7), which contains a G to C match, and #1090 (SEQ ID NO: 8), which contains a G-G mismatch, were compared to the melting behaviors of probes containing universal and generic bases. The universal or generic base containing probes analyzed included 5′ OGX1 (SEQ ID NO: 3), 5′OGX2 (SEQ ID NO: 4), and 5′ OGX5 (SEQ ID NO: 6).
A polyacrylamide gel bandshift experiment was then conducted as follows. The gel matrix was 20% acrylamide (19:1 acrylamide to bis-acrylamide) in 1×TBE buffer and “extra” salts: 20 mM Tris-HCl, pH=7.5 at 20° C., 100 mM KCl, 10 mM MgCl2, 0.05 mM DTT, 2.5% w/v sucrose. Oligonucleotide mixtures were at approximately 5 micromolar each in formamide/dye sample buffer plus 2× of the extra salt concentrations in the acrylamide gel mixture. The gel was run in 1×TBE at 93V (19 mA) and the buffer and gel temperatures were kept stable at 26° C. during the entire electrophoretic run.
The polyacrylamide gel was scanned, lanes 1-12, and the oligonucleotide probe/DNA target sequences were analyzed. Probe and DNA target designations are provided in TABLE 3. Lanes 11 and 12 of the gel marked the position of unbound target DNAs (#1088, perfect match and #1090, single base mismatch, respectively).
Lanes 1, 2, 3, and 4 of the gel showed that the all-natural-base probes (OGC1 and OGC2) could not distinguish the single base mismatch target (#1090, lanes 2 and 4) from the perfectly matched target (#1088, lanes 1 and 3). Lanes 5 through 10, on the other hand, graphically revealed the ability of the probes containing juxtaposed universal bases to detect a single-base-mismatch under these conditions. Thus, the results above provide more evidence that antisense oligonucleotides comprising juxtaposed universal bases are more specific for a target than conventional oligonucleotides, which translates into improved antisense inhibition.










TABLE 3






Size
Name
Identity







Control Oligonucleotides:





5′ ctGctaactgagcacAggatg (C6-NH2)
21 mer
OGC1
control




(SEQ ID NO: 1)



 


5′ gagctGctaactgagcacAgg (C6-NH2)
21 mer
OGC2
control




(SEQ ID NO: 2)



 


Experimental Oligonucleotides





5′ ctGctaBBBBBgcacAggatg (C6-NH2)
21 mer
OGX1
 6/5/10




(SEQ ID NO: 3)



 


5′ gagctGctaaBBBBBcacAgg (C6-NH2)
21 mer
OGX2
10/5/6 




(SEQ ID NO: 4)



 


5′ gctGctaBBBBBgcacAgg (C6-NH2)
19 mer
OGX3





(SEQ ID NO: 5)



 


5′ gagctGctBBBBBagcacAgg (C6-NH2)
21 mer
OGX5
8/5/8




(SEQ ID NO: 6)



 


Target DNA's





3′ tactcgaCgattgactcgtgTcctactggaccctggg
#1088
Target
37 mer




(SEQ ID NO: 7)



 


3′ tactcgaGgattgactcgtgTcctactggaccctggg
#1090
Target
37 mer




(SEQ ID NO: 8)









The next example demonstrates that oligonucleotides that have a total base composition of greater than 20% universal or generic bases, wherein two or more of the universal or generic bases are in a juxtaposed position, facilitate the identification of a single base mismatch on a target, as compared to oligonucleotides having a total base composition that is less than 20% and/or no juxtaposed universal or generic bases.
EXAMPLE 6
This example provides evidence that the oligonucleotides described herein unexpectedly facilitate the identification of mutations or polymorphisms, as compared to other types of oligonucleotides, which may have universal or generic bases that are not juxtaposed. Melting behavior experiments are performed to analyze the melting behaviors of oligonucleotides that have a universal or generic base composition that is greater than or less than 20% of the total base composition. Additionally, the melting behavior effect of the universal or generic bases in a juxtaposed or non-juxtaposed position is also analyzed.
The melting temperature determinations were performed for each probe/target combination as above. All mixtures were heated to 85-95° C. for 10-15 minutes and allowed to cool to room temperature before use. Melting temperatures were determined by UV absorbence in sealed quartz cuvettes using a Varian Cary 3E UV-Visible Spectrophotometer with a Varian Cary temperature controller, controlled with Cary 01.01(4) Thermal software. Temperature gradients were decreased from 85° C. to 25° C. at 1° C. per minute.
The following oligonucleotides were analyzed: The improved probes (#3192-3187) contained increasing numbers of 5-nitroindole substitutions (B). These were compared to an all-natural base probe (3179) for the ability to distinguish between a wild-type target (3221) and a mutant target containing a single SNP (3222). Probe/target melting temperatures were extimated by thermal denaturation profiles according to previously described methods. The results revealed that a single unnatural base substitution offers lower discrimination than larger numbers of substitutions, and that unnatural base substitutions can be very effective immediately adjacent to the SNP position. See Table 4:











TABLE 4









Tm Wild Type
Tm Mutant
Delta




3211
3222
Tm


Probe

Tm ° C.
Tm ° C.
° C.





3179
Tacgtgccaggtggagcacccag
82.1
77.2
4.9



(SEQ ID NO: 8)





 


3192
TacgtgccaggBggagcacccag
79.3
73.8
5.5



(SEQ ID NO: 9)





 


3191
tacgtgccagBBBgagcacccag
74.7
68.8
5.9



(SEQ ID NO: 10)





 


3190
tacgtgccaBBBBBagcacccag
66.0
57.1
8.9



(SEQ ID NO: 11)





 


3189
tacgtgccBBBBBBBgcacccag
59.3
44.8
14.5



(SEQ ID NO: 12)





 


3188
tacgtgcBBBBBBBBBcacccag
64.6
52.0
12.6



(SEQ ID NO: 13)





 


3187
tacgtgBBBBBBBBBBBacccag
66.5
54.5
12.0



(SEQ ID NO: 14)












3221
5′ gcctgggtgctccacctggcacgtatatc 3′
Wild Type “C” target



(SEQ ID NO: 15)




                       x



3222
5′ gcctgggtgctccacctggtacgtatatc 3′
Mutation “T” target



(SEQ ID NO: 16)









Further oligonucleotides are analyzed as follows: the “Z” represents a 3-nitropyrrole at the indicated position, “G” indicates the polymorphism on TARGETS A and B, and TARGET C does not have a polymorphism. Oligonucleotides 1-4 and TARGETS A, B, and C are described in U.S. Pat. No. 5,780,233 to Guo et al., herein expressly incorporated by reference in its entirety.










(1)




5′ CTCTTGAGAGAGCTAGTATCT 3′
(SEQ ID NO: 17)


 



(2)




5′ CTCTTGZGAGAGCTZGTATCT 3′
(SEQ ID NO: 18)


 



(3)




5′ CTCTTZAGAGAGCTAZTATCT 3′
(SEQ ID NO: 19)


 



(4)




5′ CTCTZGAGAGAGCTAGZATCT 3′
(SEQ ID NO: 20)


 



(5)




5′ CTCTZZAGAGAGCTAZZZTCT 3′
(SEQ ID NO: 21)


 



(6)




5′ CTCTZZZGAGAGCTAZZATCT 3′
(SEQ ID NO: 22)


 



(7)




5′ CTCTTGAGAGAGCZZZZZTCT 3′
(SEQ ID NO: 23)


 



(8)




5′ CTCZZZZZAGAGCTAGTATCT 3′
(SEQ ID NO: 24)


 



(9)




5′ CTCZZZZZAGAGCZZGTATCT 3′
(SEQ ID NO: 25)


 



(10)




5′ CTCZZZZGAGAGCZZZTATCT 3′
(SEQ ID NO: 26)


 



(TARGET A)




AGATACTAGCGCTCTCAAGAG
(SEQ ID NO: 27)


 



(TARGET B)




AGATACTAGCTCGCTCAAGAG
(SEQ ID NO: 28)


 



(TARGET C)




AGATACTAGCTCTCTCAAGAG
(SEQ ID NO: 29)






The results will show that oligonucleotides, which have a universal or generic base composition that is greater than 20%, wherein said universal or generic bases are juxtaposed, melt from TARGETS A and B at a lower temperature than the other oligonucleotides (i.e., oligonucleotides 1-4).
The next example also demonstrates that oligonucleotides that have a total base composition of greater than 20% universal or generic bases, wherein two or more of the universal or generic bases are in a juxtaposed position, facilitate the identification of a single base mismatch on a target, as compared to oligonucleotides having a total base composition that is less than 20% and/or no juxtaposed universal or generic bases.
EXAMPLE 7
This example provides evidence that the oligonucleotides described herein facilitate the identification of mutations or polymorphisms, as compared to other types of oligonucleotides, which may have universal or generic bases that are not juxtaposed. In brief, a polyacrylamide gel band shift experiment is performed to analyze the melting behaviors of oligonucleotides that have a universal or generic base composition that is greater than or less than 20% of the total base composition. Additionally, the melting behavior effect of the universal or generic bases in a juxtaposed or non-juxtaposed position is also analyzed.
The polyacrylamide gel bandshift experiment is conducted as above. The gel matrix is 20% acrylamide (19:1 acrylamide to bis-acrylamide) in 1×TBE buffer and “extra” salts: 20 mM Tris-HCl, pH=7.5 at 20° C., 100 mM KCl, 10 mM MgCl2, 0.05 mM DTT, 2.5% w/v sucrose. Oligonucleotide mixtures are at approximately 5 micromolar each in formamide/dye sample buffer plus 2× of the extra salt concentrations in the acrylamide gel mixture. The gel is run in 1×TBE at 93V (19 mA) and the buffer and gel temperatures are kept stable at 26° C. during the entire electrophoretic run.
The following oligonucleotides are analyzed; the “Z” represents a 3-nitropyrrole at the indicated position, “G” indicates the polymorphism on TARGETS A and B, and TARGET C does not have a polymorphism. Oligonucleotides 1-4 and TARGETS A, B, and C are described in U.S. Pat. No. 5,780,233 to Guo et al., herein expressly incorporated by reference in its entirety.










(1)




5′ CTCTTGAGAGAGCTAGTATCT 3′
(SEQ ID NO: 30)


 



(2)




5′ CTCTTGZGAGAGCTZGTATCT 3′
(SEQ ID NO: 31)


 



(3)




5′ CTCTTZAGAGAGCTAZTATCT 3′
(SEQ ID NO: 32)


 



(4)




5′ CTCTZGAGAGAGCTAGZATCT 3′
(SEQ ID NO: 33)


 



(5)




5′ CTCTZZAGAGAGCTAZZZTCT 3′
(SEQ ID NO: 34)


 



(6)




5′ CTCTZZZGAGAGCTAZZATCT 3′
(SEQ ID NO: 35)


 



(7)




5′ CTCTTGAGAGAGCZZZZZTCT 3′
(SEQ ID NO: 36)


 



(8)




5′ CTCZZZZZAGAGCTAGTATCT 3′
(SEQ ID NO: 37)


 



(9)




5′ CTCZZZZZAGAGCZZGTATCT 3′
(SEQ ID NO: 38)


 



(10)




5′ CTCZZZZGAGAGCZZZTATCT 3′
(SEQ ID NO: 39)


 



(TARGET A)




AGATACTAGCGCTCTCAAGAG
(SEQ ID NO: 40)


 



(TARGET B)




AGATACTAGCTCGCTCAAGAG
(SEQ ID NO: 41)


 



(TARGET C)




AGATACTAGCTCTCTCAAGAG
(SEQ ID NO: 42)






The gel will reveal that oligonucleotides, which have a universal or generic base composition that is greater than 20%, wherein said universal or generic bases are juxtaposed, facilitate the identification of the mismatch in TARGETS A and B, as compared to the other oligonucleotides (i.e., oligonucleotides 1-4).
The next example describes the use of the oligonucleotides described herein in conjunction with the TaqmanTM assay to identify a SNP associated with Hereditary hemochromatosis.
EXAMPLE 8
TaqmanTM probes derive their fluorescence signal from the hydrolysis of the probe by Taq's 5′ to 3′ exonuclease activity. The hydrolysis separates fluorescein from a quenching dye and results in an increased fluorescein signal. These probes can be used in the LightCyclerTM and are monitored in F1 or F1/F2. In the following assay, the C282Y mutation in the Hereditary hemochromatosis (HH) gene (HFE) is assayed using dried blood spots from a number of neonatal samples of dried blood.
The C282Y mutation is determined by the TaqManTM technology, which is based on the use of two fluorescent dyes (a reporter and a quencher), both attached to the probe. During PCR, the probe anneals to the target sequence between the forward and the reverse primer sites. If hybridization occurs, the probe is cleaved by the 5′-nuclease activity of the polymerase. This separates the reporter from the quencher, generating an increase in the reporter's fluorescence. Differences in fluorescence facilitate discrimination of all HFE genotypes at the 282 position. The TaqmanTM method is described in Kazuko, et al. U.S. Pat. No. 5,952,202, issued Sep. 14, 1999 (herein expressly incorporated by reference in its entirety).
PCR is performed by amplifying 100-500 ng of genomic DNA in a 50-μl volume using the TaqMan PCR Core Reagent Kit reagents (PE Applied Biosystems, Foster City, Calif.). The following conditions are used: 8% (vol/vol) glycerol, 1× TaqMan buffer, 5 mM MgCl2, 200 μM dNTP mix, 300 nM of both primers, 200 nM of normal probe, 50 nM of mutated probe, 0.01 U/μl Amp Erase UNG, and 0.05 U.μl AmpliTaq Gold. However, primers are modified, as disclosed above, for comparison.
After an incubation of 2 min at 50° C. for optimal activity of AmpErase Uracil-N-Glycosylase, and an incubation of 10 min. at 95° C. to activate AmpliTaq Gold, the following cycling protocol is run: 40 cycles of denaturation at 95° C. for 15 sec, then annealing and extension at 64° C. for 1 min. Reactions are performed in an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif.). Fluorescence is measured directly in the closed tubes. Also included in the assay are control reactions containing no template, known allele:template 1 (homozygous mutant), and known allele:template 2 (homozygous normal).
The primers modified as in the preferred embodiments described herein are able to identify the mismatch with a much lower number of false negatives and false positives than conventional probes. Accordingly, the embodiments described herein facilitate the discrimination of an SNP using the TaqmanTM assay. The next example describes the use of the oligonucleotides described herein in conjunction with the HybProbeTM assay to identify a SNP associated with vascular disease.
EXAMPLE 9
The HybProbeTM assay is also called a two-color melting curve assay. HybProbeTM chemistry consists in two adjacent probes in a head-to-tail orientation, spaced by one to four nucleotides. The probes hybridize to adjacent sequences. One of the probes is labeled at its 3′ end by a donor dye (generally flurescein). The other probe is labeled with an acceptor molecule at its 5′ end (generally LC Red640 or 705), and is phosphate-blocked at the 3′ end. When both probes are hybridized to their target sequences, the emitted light of the donor is transmitted to the acceptor fluorophore by Förster resonance energy transfer (FRET), and the Red640 emitted fluorescence (640 nm) can be detected. The intensity of the emitted fluorescence increases in parallel with the target DNA formed in the PCR. The LightCycler probes offer the advantage over the TaqManTM probe of not requiring hydrolysis and, therefore, no additional extension of the PCR times (annealing-elongation≦12 s). A method such as that in US patent applications: Wittwer et al. U.S. Pat. No. 6,232,079, issued May 15, 2001 and Wittwer et al. U.S. Pat. No. 6,140,054, issued Oct. 31, 2000 (both of which are herein incorporated by reference in their entireties) is used.
Similar to the experiments described in example 7 of the Wittwer et al. U.S. Pat. No. 6,232,079, issued May 15, 2001, the MTHFR SNP can be detected with the primers of the preferred embodiment in conjunction with a typical HybProbeTM assay. There is a common point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene (C.sub.677 T) that converts an alanine to a valine residue and results in a thermolabile enzyme. This mutation can reduce MTHFR activity and lead to elevated homocysteine plasma levels which has been implicated as an independent risk factor for early vascular disease and thrombosis as is well known in the art.
Accordingly, one of the primers is labeled with Cy5 (TGAAGGAGAAGGTGTCT*GCGGGA) (SEQ ID NO: 43) where T* represents a modified T residue linked to Cy5. The probe sequence is fluorescein-CCTCGGCTAAATAGTAGTGCGTCGA (SEQ ID NO: 44) and the other primer is AGGACGGTGCGGTGAGAGTG (SEQ ID NO: 45). Two primers and a probe are developed to conform to the primers of the preferred embodiment by making variations of SEQ ID NOS: 43 and 45.
A 198 base pair fragment of the MTHFR gene is amplified from 50 ng of human genomic DNA in 50 mM Tris, pH 8.3, 2 mM MgCl2, 500 μg/ml bovine serum albumin, 0.2 mM of each dNTP, 0.5 μM of the Cy5-labeled primer, 0.1 μM of the opposing primer, 0.1 μM of the fluorescein-labeled probe, and 0.4 U Taq DNA polymerase per 10 μl. Each cycle is 30 sec long and consisted of denaturation at 94° C. followed by a 20 sec combined annealing/extension step at 60° C. The temperature transition rate between steps is 20° C./sec. After 60 cycles, a melting curve is acquired as follows: heating from 50-65° C. at 0.5° C./sec, 65-75° C. at 0.1° C./sec, and 75-94° C. at 0.5° C./sec. After baseline subtraction and conversion to melting peaks, all possible genotypes are more easily distinguished using the primers and/or probes of the preferred embodiment, compared to the published method. The next example describes the use of the oligonucleotides described herein in conjunction with a single melting curve assay to identify a SNP associated with Hereditary hemochromatosis.
EXAMPLE 10
Minor Groove binders are dsDNA-binding dyes. They are thought to bind to the minor groove of dsDNA and upon binding increase their fluorescence over a hundred fold. An example of such a dye is SYBR Green ITM. These dyes are compatible with PCR up to a point, but at very high concentrations may start to inhibit the PCR reaction. SYBR can be used with the LightCyclerTM in channel F1. An important quality of these dyes is that they bind to any dsDNA. The specific product, non-specific products, and primer dimers are detected equally well. Therefore, it is important that the PCR reaction is extremely “clean” and does not contain any dimers or non-specific products. The primers of the preferred embodiment can be used in any PCR reaction with a minor groove binder to make the method more specific. The embodied method is as described in Kutyavin, et al. U.S. Pat. No. 6,084,102, issued Jul. 4, 2000 (herein expressly incorporated by reference) except that the oligonucleotides described herein are used.
In particular, a single melting curve assay such as that in the reference, Donohue et al. Clin Chem 2000; 46:1540-7 is used for the rapid detection of the HFE C282Y mutation that utilizes real-time multiplex, allele-specific PCR and melting curves but requires neither fluorescent hybridization probes nor any postamplification manual processing. In particular, this C282Y genotyping assay requires the design of two unlabeled allele-specific sense-strand PCR primers (and a common antisense primer) to specifically amplify (in the same tube) either the C282 or Y282 allele (or both, in the case of heterozygotes).
Nonspecific amplification of the “other” allele is prevented by several deliberate nucleotide mismatches in both allele-specific primers. During and after PCR amplification in a thermal cycler with real-time fluorescent monitoring capabilities, the amplicons are detected fluorescently, not by expensive fluorescently labeled probes, but by a nonspecific double-stranded DNA binding dye (SYBR Green I) included in the reaction. Because one of the allele-specific primers is engineered with a long 5′ GC tail to increase the melting temperature of its PCR product, the mutant- and wild-type-specific amplicons are discriminated by a progressive post-PCR temperature surge (with continuous fluorescence monitoring) to generate melting curves with characteristic allele-specific melting temperatures. The example below describes in greater detail the preparation of chips or arrays containing the oligonucleotides described herein.
EXAMPLE 11
The oligonucleotide probes described herein are particularly adaptable to current state-of-the-art chip based hybridization technologies. For example, for detection of the HMF mutation, a chip is prepared containing nucleic acids isolated from the blood samples taken from a large number of patients. Probes that are manufactured in accordance with the teachings herein are then used to detect either the C282 or Y282 allele or both. Accordingly, those individuals that have the HMF mutation are identified by virtue of the hybridization of the probe to the target sequence. In the same way, individuals who are wildtype may be identified by the presence of the wildtype allele or by the absence of a mutant allele.
Alternatively, an array can be prepared with the oligonucleotides of the present invention. A typical conventional array may contain 8-16 different oligonucleotides from each gene, for example. By using the oligonucleotides of the present invention, however, chips can be made using only 4 different oligonucleotides from each gene, thereby reducing the cost of analysis.
EXAMPLE 12
An example showing that when using Table 1, 2-Thio-dT offers better discrimination between A and G than the natural base T.
The sequences are derivatives of from Guo et al. U.S. Pat. No. 5,780,233. Oasis 3354 is identical to Guo SEQ. ID NO: 4 (identified herein as SEQ ID NO: 38). Oasis 3353 is a single 2-thio-dT substitution (upper case T) into Guo SEQ. ID NO: 4 (identified herein as SEQ ID NO: 39). Oasis 3355 is identical to Guo SEQ. ID NO: 5, but written below in the 5′-3-orientation (identified herein as SEQ ID NO: 40). Oasis 3356 contains a single mutation of G substituted for A (bold letters) (identified herein as SEQ ID NO: 41). All molecules are DNA:











3353
5′tggTtatagaagtat
15 mer 2-thio-T probe
(SEQ ID NO: 46)



 


3354
5′tggttatagaagtat
15 mer unmod. Probe
(SEQ ID NO: 47)


 


3355
5′agatacttctataaccaagag
wt target
(SEQ ID NO: 48)


 


3356
5′agatacttctatagccaagag
mut target
(SEQ ID NO: 49)






These molecules were used in optical melting experiments in the following buffer: 1×SSCKM (150 NaCl, 25 mM NaCitrate, 80 mM KCl and 10 mM MgCl2, pH=7.4 at 20° C.).
Melting temperature determination (Tm° C.) results:















3353/3355 (2-thio-T probe/wt A target)
Tm = 46.8



3354/3355 (unmod T probe/wt A target)
Tm = 46.7









Accordingly, under these buffer conditions 2-thio-dT does not bind dA more or less tightly than dT as shown by the equivalent melting temperatures.
Melting temperature determination (Tm° C.) results:















3353/3356 (2-thio-T probe/mut G target)
Tm = 40.7



3354/3356 (unmod T probe/mut G target)
Tm = 42.6









Thus, under these buffer conditions a 2-thio-dT mismatch to dG is more discriminatory than a natural dT:dG mismatch, resulting in a melting temperature 2° C. lower. For this reason, a 2-thio-dT-containing probe has greater sequence specificity and greater allele specificity.
The utility of this observation in probe design is born out by the following comparison of the same data:
Melting temperature determination (Tm° C.) results:















3353/3355 (2-thio-T probe/wt A target)
Tm = 46.8



3353/3356 (2-thio-T probe/mut G target)
Tm = 40.7




ΔTm = 6° C.



3354/3355 (unmod T probe/wt A target)
Tm = 46.7



3354/3356 (unmod T probe/mut G target)
Tm = 42.6




ΔTm = 4° C.









One can readily see that a probe containing a 2-thio-dT substitution in the SNP position yields greater discrimination between two targets containing A versus G (ΔTm=6° C.) than natural dT (ΔTm=4° C.). An example of choosing an unnatural base from Table 1 for a G to A SNP is shown in Example 13.
EXAMPLE 13
The human hemochromatosis gene (HFE, accession no. XM—004413.3) is a clinically important gene and several research groups have published diagnostic assay procedures hoping to establish routine clinical screening for the presence of mutated HFE genes in the general population. Mutations in HFE cause iron accumulation that can lead to serious illness and even death.
One of the most frequent mutations is a G to A base change (G845A at position 1066). If the wild type and mutant alleles need to be detected, or diagnosed, on the basis of the SNP at position 1066 (shown below), the SNP Discrimination Table can be used to design more discriminatory probes than those based on natural bases alone (where C=5-methyl-dC and T=2-thio-dT).








Wild type allele (5′-3′):











1041
cctggggaag agcagagata tacgtgccag gtggagcacc caggcctgga
(SEQ ID NO: 50)



 








Mutant allele (5′-3′):











1041
cctggggaag agcagagata tacgtaccag gtggagcacc caggcctgga
(SEQ ID NO: 51)







Probe Design to Detect the Wild Type Allele.

The natural base to bind is G and the natural base to not bind is A, thus we have the following evolution of probe sequence selections:










5′ c ctggcacgta tatctctgct ct
conventional, low disc.
(SEQ ID NO: 52)



 


5′ c ctggcacgtB BBBBtctgct ct
Universal base
(SEQ ID NO: 53)


 


5′ c ctggCacgtB BBBBtctgct ct
UB with SNP disc.
(SEQ ID NO: 54)






Probe Design to Detect the Mutant Allele

The natural base to bind is A and the natural base to not bind is G, thus we have the following evolution of probes:










5′ c ctggtacgta tatctctgct ct
conventional, low disc.
(SEQ ID NO: 55)



 


5′ c ctggtacgtB BBBBtctgct ct
Universal base
(SEQ ID NO: 56)


 


5′ c ctggTacgtB BBBBtctgct ct
UB with SNP disc.
(SEQ ID NO: 57)






The use of the 5-methyl-dC and 2-thio-dT come directly from looking at the table axes as they are labeled, which natural bases to bind versus which natural bases to avoid. The combination of the non-hydrogen bonding universal bases with the unnatural bases in the SNP position should produce probes far superior to the conventional, low discrimination probes.
The following example demonstrates the use of oligonucleotides having juxtaposed universal bases to increase specificity for a target sequence.
EXAMPLE 14
Two sets of probes were used to detect polymorphisms in target DNA sequences. A first “improved” set of probes included oligonucleotides containing universal bases. A second “natural” set of probes contained only naturally occurring bases. Each set of immobilized probe pairs was used to detect single nucleotide polymorphisms (SNPs) in fluorescently labeled target oligonucleotides.
Natural probe pairs and Improved probe pairs consist of one probe oligonucleotide which formed perfect base-pairing, and one probe which contained one mismatched base-pairing when hybridized to the target oligonucleotide. The Improved probe pairs contained a variable number of artificial base mismatches using base analogues other than G, A, T, U or C such as generic, universal or degenerate bases.
The Improved probes and natural probes used in the microarray analysis were 5′-amine modified oligonucleotides. They were prepared to a final concentration of 30 uM in array printing buffer (150 mM sodium phosphate, pH8.5), and printed on to 3D-Link activated slides (Motorola Life Sciences, product # DN01-0025) by using a manual glass slide microarrayer system with a floating pin replicator (VP-scientific, product # VP478) and a glass slide indexer (VP-scientific, product # VP470) used according to the arrayer instructions.
After printing, slides were coupled and blocked according to the glass slide manufacture's recommended procedure (Motorola Life Sciences, product # DN01005). Briefly, freshly printed slides were incubated in a saturated NaCl chamber overnight to allow the 5′-amine-oligonucleotides to conjugate to the slide surface. Excess reactive surface sights were blocked after conjugation with pre-warmed blocking solution (0.1 M Tris, 50 mM ethanolamine, 0.1% SDS, pH 9.0) at 50° C. for 15 minutes, followed by a 4×SSC/0.1% SDS wash for 60 minutes.
The targets used in the microarray assays were 5′-fluorescent labeled oligonucleotides that contained single nucleotide polymorphism (SNP) sites. They were prepared to a final concentration of 10 uM in hybridization solution (5×SSC, 0.1 mg/ml salmon sperm DNA, 0.1% SDS or 0.05% Triton X-100). A cover slide (LifterSlip by Erie Scientific Co., product #22IX25-2-4635) was used to cover the array area on the slide and hold  ̃20 ul of hybridization solution.
The whole slide set was then kept in a hybridization cassette (TeleChem, product # AHC) for 2 hrs at various temperature (ranging from room temperature to 50° C.) followed by a 10-minute wash with washing solution (2×, 4× or 5×SSC, 0.1% SDS or 0.05% TritonX-100) at the hybridization temperature. The fluorescent signal on the slide was monitored by a fluorescent microscope (Nikon, Labophot-2), and the image was taken by digital camera (CoolSnap HQ) and analyzed by the MetaMorph image analysis program (Universal Imaging Corporation).
Immobilized Improved probes and natural probes were evaluated for hybridization between three SNP target fragment oligonucleotides from the human hemochromatosis gene (SNPs A, B and C). The hybridization specificity is presented as the ratio of perfect match (abbreviated as ‘pm’) to mismatch (abbreviated as ‘mm’) hybridization.
The oligonucleotide sequences of the target mimics and probes used are listed below. The SNP site of each oligo is underlined.
The following are target oligonucleotides with a phosphodiester backbone and FAM (abbreviated as ‘F’) label at the 5′ terminus.










#3533:
5′ F-CAGGCCTGGGTGCTCCACCTGGCACGTATATCTCTGCTC 3′
(SEQ ID NO: 58)



 


#3534:
5′ F-CAGGCCTGGGTGCTCCACCTGGTACGTATATCTCTGCTC 3′
(SEQ ID NO: 59)


 


#3535:
5′ F-GTTCGGGGCTCCACACGGCGACTCTCATGATCATAGAAC 3′
(SEQ ID NO: 60)


 


#3536:
5′ F-GTTCGGGGCTCCACACGGCGACACTCATGATCATAGAAC 3′
(SEQ ID NO: 61)


 


#3537:
5′ F-GGCTCCACACGGCGACTCTCATGATCATAGAACACGAACA 3′
(SEQ ID NO: 62)


 


#3538:
5′ F-GGCTCCACACGGCGACTCTCATCATCATAGAACACGAACA 3′
(SEQ ID NO: 63)






The following are natural oligonucleotide probes with a phosphodiester backbone and C6-amino linker (abbreviated as ‘NH2-C6’) followed by a spacer of four hexaethylene glycol molecules (PEG-282, abbreviated as ‘S18’) at the 5′ terminus.










#3569:
5′ NH2-C6-S18-S18-S18-S18-TATACGTGCCGGTGG 3′
(SEQ ID NO: 64)



 


#3570:
5′ NH2-C6-S18-S18-S18-S18-TATACGTACCGGTGG 3′
(SEQ ID NO: 65))


 


#3571:
5′ NH2-C6-S18-S18-S18-S18-GATCATGAGAGTCGCCGTG 3′
(SEQ ID NO: 66)


 


#3572:
5′ NH2-C6-S18-S18-S18-S18-GATCATGAGTGTCGCCGTG 3′
(SEQ ID NO: 67)


 


#3573:
5′ NH2-C6-S18-S18-S18-S18-TTCTATGATCATGAGAGTC 3′
(SEQ ID NO: 68)


 


#3574:
5′ NH2-C6-S18-S18-S18-S18-TTCTATGATGATGAGAGTC 3′
(SEQ ID NO: 69)






The following are improved probes with a phosphodiester backbone and C6-amino linker (abbreviated as ‘NH2-C6’) followed by either a spacer of four hexaethylene glycol (PEG-282, abbreviated as ‘S18’) residues or twelve deoxythymidine (abbreviated as ‘T12’) residues at the 5′ terminus. The nucleotides with universal bases used in probes are 5′-nitroindole-2′-deoxyriboside (abbreviated as ‘B’), 3-nitropyrrole-2′-deoxyriboside (abbreviated as ‘M’), 7-deaza-2′-deoxyadenosine (abbreviated as ‘A7’), 2-amino-2′-deoxyadenosine (abbreviated as ‘A2’), 2-thiothymidine (abbreviated as ‘T2’).










#3419:
5′ NH2-C6-T12-TACGTGCCBBBBBGAGCACCC 3′
(SEQ ID NO: 70)



 


#3420:
5′ NH2-C6-T12-TACGTA7CCBBBBBGAGCACCC 3′
(SEQ ID NO: 71)


 


#3421:
5′ NH2-C6-T12-TACGTGCCBBBBBGAGCACC 3′
(SEQ ID NO: 72)


 


#3422:
5′ NH2-C6-T12-TACGTA7CCBBBBBGAGCACC 3′
(SEQ ID NO: 73)


 


#3575:
5′ NH2-C6-S18-S18-S18-S18-TACGTGCCBBBBBGAGCACC 3′
(SEQ ID NO: 74)


 


#3576:
5′ NH2-C6-S18-S18-S18-S18-TACGTACCBBBBBGAGCACC 3′
(SEQ ID NO: 75)


 


#3581:
5′ NH2-C6-S18-S18-S18-S18-TACGTGCCBBBMBGAGCACC 3′
(SEQ ID NO: 76)


 


#3582:
5′ NH2-C6-S18-S18-S18-S18-TACGTACCBBBMBGAGCACC 3′
(SEQ ID NO: 77)


 


#3423:
5′ NH2-C6-T12-ATGAGA2GTBBBBBTGTGGAGC 3′
(SEQ ID NO: 78)


 


#3424:
5′ NH2-C6-T12-ATGAGT2GTBBBBBTGTGGAGC 3′
(SEQ ID NO: 79)


 


#3425:
5′ NH2-C6-T12-CTATGABBBBBAGA2GTBBBBBTGTGGA 3′
(SEQ ID NO: 80)


 


#3426:
5′ NH2-C6-T12-CTATGABBBBBAGT2GTBBBBBTGTGGA 3′
(SEQ ID NO: 81)


 


#3589:
5′ NH2-C6-S18-S18-S18-S18-TTCTATGABBBBBAGA2GTBBBBBTGTGGAGC 3′
(SEQ ID NO: 82)


 


#3590:
5′ NH2-C6-S18-S18-S18-S18-TTCTATGABBBBBAGT2GTBBBBBTGTGGAGC 3′
(SEQ ID NO: 83)


 


#3591:
5′ NH2-C6-S18-S18-S18-S18-TCGTGTTBBBBBATCATGAG 3′
(SEQ ID NO: 84)


 


#3592:
5′ NH2-C6-S18-S18-S18-S18-TCGTGTTBBBBBATGATGAG 3′
(SEQ ID NO: 85)






The following are improved probes with a hybrid backbone of phosphodiester and 2′-O-Methyl-RNA (abbreviated as ‘No’).










#3539:
5′ NH2-C6-S18-S18-S18-S18-U0A0C0G0U0GCCBBBBBGAGCACC 3′
(SEQ ID NO: 86)



 


#3540:
5′ NH2-C6-S18-S18-S18-S18-U0A0C0G0U0ACCBBBBBGAGCACC 3′
(SEQ ID NO: 87)


 


#3521:
5′ NH2-C6-S18-S18-S18-S18-TACGTGCCBBBBBG0A0G0C0A0C0C0C0 3′
(SEQ ID NO: 88)


 


#3522:
5′ NH2-C6-S18-S18-S18-S18-TACGTACCBBBBBG0A0G0C0A0C0C0C0 3′
(SEQ ID NO: 89)


 


#3541:
5′ NH2-C6-S18-S18-S18-S18-A0U0G0A0G0A2GTBBBBBTGTGGAGCA 3′
(SEQ ID NO: 90)


 


#3542:
5′ NH2-C6-S18-S18-S18-S18-A0U0G0A0G0T2GTBBBBBTGTGGAGC 3′
(SEQ ID NO: 91)


 


#3529:
5′ NH2-C6-S18-S18-S18-S18-ATGATCATBBBBBBC0G0C0C0G0U0G0U0 3′
(SEQ ID NO: 92)


 


#3530:
5′ NH2-C6-S18-S18-S18-S18-ATGATGATBBBBBBC0G0C0C0G0U0G0U0 3′
(SEQ ID NO: 93)






The results of the hybridization specificity of natural probes and Improved probes are listed in Table 5.












TABLE 5





Probe
#
#
Specificity
Condition
Salt in


Type
pm
mm
Ratio
Temperature
washing















site A, wild type target (#3533)












natural
3569
3570
5
50° C.
2X SSC


Improved probe
3539
3540
10
R° T
5X SSC







site A, mutant target (#3534)












natural
3570
3569
1.5
50° C.
2X SSC


Improved probe
3540
3539
30
45° C.
2X SSC


Improved probe
3522
3521
30
45° C.
2X SSC


Improved probe
3420
3419
15
45° C.
2X SSC


Improved probe
3422
3421
18
45° C.
2X SSC


Improved probe
3582
3581
23
37° C.
5X SSC


Improved probe
3576
3575
15
30° C.
5X SSC







site B, wild type target (#3535)












natural
3571
3572
1
50° C.
2-5X SSC


Improved probe
3541
3542
22
45° C.
2X SSC


Improved probe
3541
3542
15
30° C.
2X SSC


Improved probe
3523
3524
6
30° C.
2X SSC


Improved probe
3525
3526
14
37° C.
4X SSC


Improved probe
3589
3590
3
37° C.
5X SSC







site B, mutant target (#3536)












natural
3572
3571
1
50° C.
2-5X SSC


Improved probe
3542
3541
54
45° C.
4X SSC


Improved probe
3524
3523
3
45° C.
4X SSC


Improved probe
3426
3425
8
45° C.
2X SSC


Improved probe
3426
3425
5
30° C.
5X SSC


Improved probe
3590
3589
5
37° C.
5X SSC







site C, wild type target (#3537)












natural
3573
3574
9
50° C.
2X SSC


Improved probe
3591
3592
7
R° T
5X SSC


Improved probe
3529
3530
12
37° C.
5X SSC







site C, mutant target (#3538)












natural
3574
3573
11
50° C.
2X SSC


Improved probe
3592
3591
30
45° C.
2X SSC


Improved probe
3592
3591
45
R° T
5X SSC









The results indicated that Improved probe sets having juxtaposed universal bases had improved hybridization specificity when compared to natural probe sets in all cases. The natural probe sets used were designed with a SNP site in the middle and optimized to give the best differential hybridization between pm and mm, but were not all capable of showing good discrimination. Improved probe sets with a block of universal bases at the 5′ and/or 3′ to the SNP site, with two or more universal base blocks, and other unnatural bases, significantly increased hybridization specificity to discriminate mm from pm, even in the case where the natural probe sets showed good discrimination.
Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references including: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., (1987); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc. (1986); Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., John Wiley & Sons, (1992), Grinsted et al., Plasmid Technology, Methods in Microbiology, Vol. 21, Academic Press, Inc., (1988); Symonds et al., Phage Mu, Cold Spring Harbor Laboratory Press (1987), Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.).
The basic principles of eukaryotic gene structure and expression are generally known in the art. (See for example Hawkins, Gene Structure and Expression, Cambridge University Press, Cambridge, UK, 1985; Alberts et al., The Molecular Biology of the Cell, Garland Press, New York, 1983; Goeddel, Gene Expression Technology, Methods in Enzymology, Vol. 185, Academic Press, Inc., (1991); Lewin, Genes VI, Oxford Press, Oxford, UK, 1998). Each of the above-mentioned references are hereby incorporated by reference in their entirety.
Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. All references cited herein are hereby expressly incorporated by reference.

## Claims

1. An oligonucleotide probe for detecting a first target nucleic acid sequence comprising the formula:


XRY

wherein X comprises between about 5 and 20 modified nucleic acid bases complementary to a first region of said first target nucleic acid sequence;
wherein R comprises between about 5 and 10 universal or generic bases juxtaposed to one another and said universal or generic bases align with bases of a second region of said first target nucleic acid sequence;
wherein Y comprises between about 5 and about 20 nucleic acid bases complementary to a third region of said first target nucleic acid sequence;
wherein X, R, and Y are covalently joined;
wherein greater than 20% of the total number of bases in said oligonucleotide probe are said universal or generic bases juxtaposed to one another;
wherein said oligonucleotide probe further comprises a detectable label; and
wherein said oligonucleotide probe is capable of hybridizing to and detecting said first target nucleic acid sequence but not able to detect a second target nucleic acid sequence that differs from said first target nucleic acid sequence by a single nucleotide base mismatch in the first or third region of the target nucleic acid sequence.

2. An oligonucleotide probe for distinguishing a first target nucleic acid sequence from a second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch, said oligonucleotide probe comprising
a site complementary to said first target nucleic acid sequence, said site having a 5′ and 3′ side thereto and having a non-complementary base to said mismatch of said second target nucleic acid sequence;
at least five universal or generic bases juxtaposed to one another, said universal or generic bases being located on the 5′ or 3′ side of said site and aligned with bases of the first target nucleic acid sequence;
a first region complementary to a first region of the first target nucleic acid and a second region complementary to a second region of the first target nucleic acid, said first and second regions being located on the 5′ and 3′ side of said site, respectively;
wherein said oligonucleotide probe has between about 10 and 25 bases;
wherein said oligonucleotide probe further comprises a detectable label;
and wherein said probe detects said first target nucleic acid sequence and does not detect said second target sequence.

3. The oligonucleotide probe of claim 1, wherein at least 6 of said bases are universal or generic bases juxtaposed to one another.

4. The oligonucleotide probe of claim 1, wherein at least 7 of said bases are universal or generic bases juxtaposed to one another.

5. The oligonucleotide probe of claim 1 wherein said universal bases are selected from the group consisting of: 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, and 2-deoxynebularine.

6. The oligonucleotide probe of claim 1, wherein said oligonucleotide probe comprises between about 10 and about 25 bases.

7. The oligonucleotide probe of claim 1, wherein said oligonucleotide probe comprises between about 10 and about 50 bases.

8. The oligonucleotide probe of claim 1, further comprising a non-nucleic acid linker.

9. The oligonucleotide probe of claim 1, wherein 30% or more of said bases are universal or generic bases.

10. A hybrid consisting of the oligonucleotide probe of claim 1 and a target nucleic acid sequence.

11. The oligonucleotide probe of claim 1, wherein said oligonucleotide probe has an increased ability to distinguish said first target nucleic acid sequence from said second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch as compared to an oligonucleotide probe that does not have any universal or generic bases.

12. The oligonucleotide probe of claim 2, wherein at least 6 of said bases are universal or generic bases juxtaposed to one another.

13. The oligonucleotide probe of claim 2, wherein at least 7 of said bases are universal or generic bases juxtaposed to one another.

14. The oligonucleotide probe of claim 2 wherein said universal bases are selected from the group consisting of: 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, and 2-deoxynebularine.

15. The oligonucleotide probe of claim 2, wherein said oligonucleotide probe comprises between about 10 and about 50 bases.

16. The oligonucleotide probe of claim 2, further comprising a non-nucleic acid linker.

17. The oligonucleotide probe of claim 2, wherein greater than 20% of said bases are universal or generic bases.

18. The oligonucleotide probe of claim 2, wherein 30% or more of said bases are universal or generic bases.

19. A hybrid consisting of the oligonucleotide probe of claim 2 and a target nucleic acid sequence.

20. The oligonucleotide probe of claim 2, wherein said oligonucleotide probe has an increased ability to distinguish said first target nucleic acid sequence from said second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch as compared to an oligonucleotide probe that does not have any universal or generic bases.

21. The oligonucleotide probe of claim 1 or 2, wherein said universal bases are selected from the group consisting of: 2-deoxyinosine and 2-deoxynebularine.

22. The oligonucleotide probe of claim 1 or 2, wherein said universal bases are 3-nitropyrrole.

23. The oligonucleotide probe of claim 1 or 2, wherein said universal bases are 5-nitroindole.