(19)
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`(12)
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`Europäisches Patentamt
`
`European Patent Office
`
`Office européen des brevets
`
`*EP000946750B1*
`EP 0 946 750 B1
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`(11)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`22.08.2001 Bulletin 2001/34
`
`(21) Application number: 97949674.2
`
`(22) Date of filing: 01.12.1997
`
`(51) Int Cl.7: C12Q 1/68
`
`(86) International application number:
`PCT/US97/21845
`
`(87) International publication number:
`WO 98/24933 (11.06.1998 Gazette 1998/23)
`
`(54) METHODS, KITS AND COMPOSITIONS FOR SUPPRESSING THE BINDING OF DETECTABLE
`PROBES TO NON-TARGET SEQUENCES IN HYBRIDIZATION ASSAYS
`
`VERFAHREN, REAGENTIENSÄTZE UND ZUSAMMENSETZUNGEN ZUR UNTERDRÜCKUNG
`DER BINDUNG VON DETEKTIERBAREN SONDEN AN NICHT-ZIELSEQUENZEN IN
`HYBRIDISIERUNGSTESTS
`
`PROCEDES, KITS ET COMPOSITIONS PERMETTANT DE SUPPRIMER LA FIXATION DE
`SONDES DETECTABLES A DES SEQUENCES NON-CIBLES D’ACIDE NUCLEIQUE DANS DES
`DOSAGES D’HYBRIDATION
`
`(84) Designated Contracting States:
`DE DK FR GB
`
`(30) Priority: 04.12.1996 US 32349 P
`25.09.1997 US 937709
`03.11.1997 US 963472
`
`(43) Date of publication of application:
`06.10.1999 Bulletin 1999/40
`
`(73) Proprietors:
`• Boston Probes, Inc.
`Bedford, MA 01730 (US)
`• DAKO A/S
`2600 Glostrup (DK)
`
`(72) Inventors:
`• COULL, James, M.
`Westford, MA 01886 (US)
`• HYLDIG-NIELSEN, Jens, J.
`Holliston, MA 01746 (US)
`• GODTFREDSEN, Sven, E.
`DK-3500 Vaerlose (DK)
`• FIANDACA, Mark, J.
`Acton, MA 01720 (US)
`
`• STEFANO, Kyriaki
`Hopkinton, MA 10748 (US)
`
`(74) Representative: Christiansen, Ejvind et al
`Hofman-Bang A/S
`Hans Bekkevolds Allé 7
`2900 Hellerup (DK)
`
`(56) References cited:
`EP-A- 0 304 184
`EP-A- 0 725 148
`
`EP-A- 0 546 590
`WO-A-95/17430
`
`• EGHOLM M ET AL: "PNA HYBRIDIZES TO
`COMPLEMENTARY OLIGONUCLEOTIDES
`OBEYING THE WATSON-CRICK
`HYDROGEN-BONDING RULES" NATURE, vol.
`365, 7 October 1993, pages 566-568,
`XP002030791
`• PLUSKAL M. ET AL.: "Peptide nucleic acid
`probes and their application in DNA and RNA
`hybridisation analysis (abstract nr. 35)", THE
`FASEB JOURNAL, , 19. April 1994, vol. , no. ,
`page A1264
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
`99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
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`Description
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`Background:
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`1. Technical Field
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`[0001] This invention is related to the field of probe based nucleic acid sequence detection, quantitation and analysis.
`More specifically, this invention relates to methods, kits and compositions suitable for suppressing the binding of de-
`tectable nucleic acid probes or detectable PNA probes to non-target sequences in an assay for detecting a target
`sequence of a nucleic acid molecule of interest.
`
`2. Background Art
`
`[0002] Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes
`have long been used to analyze samples for the presence of nucleic add from a bacteria, fungi, virus or other organism
`(See for example; US patents: 4,851,330,5,288,611, 5,567,587, 5,601,984 and 5,612,183). Probe-based assays are
`also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based
`assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result
`of difficulties associated with specificity, sensitivity and reliability.
`[0003] Nucleic add hybridization is a fundamental process in molecular biology. Sequence differences as subtle as
`a single base (point mutation) in very short oligomers (< 10 base pairs "bp") can be sufficient to enable the discrimination
`of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. None-
`theless, nucleic acid probes of greater than 10 bp in length are generally required to obtain the sequence diversity
`necessary to correctly identify a unique organism, disease state or clinical condition of interest. However, the ability to
`discriminate between closely related sequences is inversely proportional to the length of the hybridization probe be-
`cause the difference in thermal stability decreases between wild type and mutant complexes as the probe length in-
`creases. Consequently, the power of probe based hybridization to correctly identify the target sequence of interest
`from closely related (e.g. point mutations) non-target sequences can be very limited.
`[0004] An extensive review of the "Prinaples and Practices of Nucleic Acid Hybridization" is available (See: David E
`Kennell, Prinaples and Practices of Nucleic Add Hybridization, pp. 259-301). In the manuscript, the author discusses
`the "Use of Competitor RNA to Estimate Specifiaty". This process is based on the principle that two identical molecules
`will compete with each other for a common binding site. This principle is applied to assess similarities between two
`RNA populations competing for a common DNA. Typically, one population of RNA is labeled and the competitor pop-
`ulation of RNA is unlabeled. The competition assay is used to estimate the degree of relation between the through the
`two RNA species. A process called "presaturation competition", wherein the unlabeled competitor RNA is hybridized
`to the DNA before hybridization of the labeled RNA, has been reported to be useful in improving the results of this type
`of assay (See: p 297). However, the author warns that "great caution should be exercised " in interpreting the data
`from these assays (See: p.291 and p. 298 first full paragraph). No data is provided which quantitates the benefits
`associated with the application of this methodology.
`[0005] Gray et al. describe in-situ methods for chromosome-spedfic staining wherein the hybridization of labeled
`nucleic acid fragments to repetitive sequences of chromosomal DNA is disabled (See: Gray et al. US Pat. No.
`5,447,841). In one embodiment of the invention, disabling of the hybridization capacity of the repetitive DNA sequences
`within nucleic acid fragments involves blocking the repetitive sequences by pre-reassociation of fragments with frag-
`ments of repetitive-sequence-rich DNA, by pre-reassociation of target DNA with fragments of repetitive-sequence-rich
`DNA, or pre-reassociation of both the fragments of the heterogeneous mixture and the target DNA with repetitive-
`sequence-rich DNA (See: col. 9, Ins. 58-68). The pre-reassociation procedure may be performed in a number of differing
`formats (See: claims 2-5). This method provides blocking sufficient to permit detection of large labeled nucleic add
`(greater than 1000 bp) hybridized to chromosomal DNA (See: Claim 1). No data is provided which quantitates the
`benefits associated with the application of this methodology. Moreover, this treatment merely results in nucleic acid
`fragments whose repetitive sequences are blocked by complementary fragments such that sufficient unique sequence
`regions remain free for attachment to chromosomal DNA during the in-situ hybridization step (See: col. 10, Ins. 3-13).
`[0006] Hybridization assays hold promise as a means to screen large numbers of patient samples for a large number
`of mutations. In practice, however, it is often difficult to multiplex an assay given the requirement that each of the many
`very different probes in the assay must exhibit a very high degree of specificity for a specific target nucleic acid under
`the same or similar conditions of stringency. Recently however, a probe based assay has been shown to be effective
`at selectively detecting up to twelve cystic fibrosis transmembrane conductance regulator (CFTR) mutations using
`pools of allele specific oligonucleotides "ASOs" (See: Shuber et al., Human Mol. Gen., (1993) 2, 153-158). The authors
`utilized a tetramethylammonium chloride (TMAC) buffer to eliminate variability in the affinity of the nudeic acid probes
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`for their complementary target nucleic acid sequences. Interestingly, the authors describe the use of labeled and un-
`labeled nucleic acid probes in the hybridization cocktail. However, there is no discussion of the rational for applying
`this methodology and there is no data provided which quantitates the benefits associated with application of this tech-
`nology.
`[0007] More recently, Shuber and his coworkers introduced a technique they coined MASDA (multiplex allele specific
`diagnostic assay). See: Shuber et al. Human Mol. Gen. (1997) 6, 337-347. In this assay, a single hybridization is
`performed with a pool of allele specific oligonucleotide probes. The ASOs are affinity purified from the pool by hybrid-
`ization to the target nucleic add (patient sample) which has been immobilized to a surface. Probes, which hybridize to
`the target nucleic acid, are thereafter eluted from the surface and analyzed to thereby determine the presence or
`absence of one or more clinical conditions of interest. The authors report that they observe such a high degree of
`specificity of hybridization of the component labeled ASOs of the pool that, in a single assay, the method is capable
`of analyzing greater than 500 samples for greater than 100 known mutations. As in the prior Shuber publication, the
`authors describe the use of a hybridization cocktail containing both labeled and unlabeled probes. This cocktail is
`prepared to achieve uniform hybridization signals in the assay. However, no data is provided which quantitates the
`benefits associated with the application of this methodology.
`[0008]
`It has been reported that oligonudeotides can be used to reduce or eliminate the hybridization of target probes
`to non-target sequences (See: Arnold, L.J. Jr.; European Patent Application, EP-A-304,184). However, despite recog-
`nizing the difficulty of nucleic acid point mutation analysis, Arnold does not expressly describe, suggest or teach that
`his invention is suitable for point mutation analysis. More importantly, the model systems used as Examples to illustrate
`his invention have a two base pair mismatch between the target probe and the non-target probe. Additionally, an
`important recognized limitation of the Arnold invention is the potential for reduction in specific signal caused by the
`cross reaction between the target sequence and non-target probe to thereby form a target sequence/non-target probe
`hybrid (See: p. 3, Ins. 27-31 and lns 48-50). This may explain why Arnold specifically expresses a preference for lower
`ratios of non-target probe to target probe (See: p. 7, Ins. 53-54). The data in Tables 1-4 clearly demonstrates that as
`the ratio of non-target probe to target probe increases, there is a substantial decrease in the specific signal.
`[0009]
`In a similar fashion, the use of "blocking oligonucleotides" to suppress the capture of amplified nucleic acids
`of non-target organisms has also been described (See: Nycz et al, European Patent Application EP-A-725,148). Unlike
`Arnold, Nycz et al. did utilize "blocking oligonucleotides" which were single point mutations of the capture probes used
`to immobilize the amplicons to a support and thereby achieve species specific detection of nucleic acid samples.
`However, Nycz et al. used capture probe to blocker probes ratios of only 1 to 1 (See: discussion of Examples 2-5).
`Like Arnold, Nycz et al. observed as much as a thirty percent reduction in specific signal in one assay (See: The
`discussion of Example 3 on page 11) and careful analysis of the data provided in the Tables likewise revealed significant
`signal reductions in almost all cases for which comparative data was shown.
`[0010] The background art thus far discussed does not disclose, suggest or teach anything about Peptide Nucleic
`Acids (PNAs).
`[0011] Peptide Nucleic Acids (PNAs) are non-naturally occurring polyamides which can hybridize to nucleic acids
`(DNA and RNA) with sequence specificity. (See United States Patent No. 5,539,082 and Egholm et al., Nature (1993)
`365, 566 568 as well as Pluskal et al., Faseb Journal, Abstract No. 35, 1994)). PNA's are candidates for investigation
`as alternatives/substitutes to nucleic acid probes in probe-based hybridization assays because they exhibit several
`desirable properties. PNA's are achiral polymers which hybridize to nucleic acids to form hybrids which are more ther-
`modynamically stable than a corresponding nucleic acid/nucleic acid complex (See: Egholm et. al., Nature (1993) 365,
`566-568 as well as Pluskal et al., Faseb Journal, Abstract No. 35, 1994)). Being non-naturally occurring molecules,
`they are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore,
`PNA's should be stable in biological samples, as well as, have a long shelf-life. Unlike nucleic acid hybridization which
`is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength
`and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic
`acid (See: Egholm et. al., Nature, p. 567). The effect of ionic strength on the stability and conformation of PNA com-
`plexes has been extensively investigated (See: Tomac et al. J. Am. Chem. Soc. (1996) 118, 5544-5552). Sequence
`discrimination is more effiaent for PNA recognizing DNA than for DNA recognizing DNA (See: Egholm et al., Nature,
`p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes,
`in a hybridization assay appears to be somewhat sequence dependent (See: Nielsen et al. Anti-Cancer Drug Design
`(1993) 8, 53 65). As an additional advantage, PNA's hybridize to nucleic add in both a parallel and antiparallel orien-
`tation, though the antiparallel orientation is preferred (See: Egholm et al., Nature, p. 566).
`[0012] PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now com-
`mercially available. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm
`et al., New J. Chem. (1997), 21, 19-31 or Hyrup et. aL, Bioorganic & Med. Chem. (1996) 4, 5-23). Labeled and unlabeled
`PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No.
`NL612, Practical PNA, Review and Practical PNA, Vol. 1, Iss. 2) or prepared using the commercially available products.
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`[0013]
`Labeled PNA probes have been hybridized to target nucleic acid subsequences of denatured dsDNA as a
`means to detect the presence and amount of the DNA of interest in an assay coined "pre-gel hybridization" (See:
`O'Keefe et al. Proc. Natl. Acad. Sci. USA (1996) 93,14670-14675). This assay relies on the rapid kinetics of PNA/DNA
`hybrid formation and the relatively slow rate of reannealing of the dsDNA. Thus, under conditions of low salt, the sample
`is analyzed for the presence of the PNA/DNA hybrid before the PNA/nucleic add complex is dissociated by the rean-
`nealing/reformation of the dsDNA. "Pre-gel hybridization" is reported to provide very good discrimination of point mu-
`tations in a DNA sample (See: figure 4 of the O'Keefe manuscript and the associated description).
`[0014]
`In a similar manner, unlabeled PNAs have been shown to be effective at blocking the interstrand and intras-
`trand interactions of dsDNA to thereby enhance the PCR amplification of variable numbers of tandem repeat (VNTR)
`loci (See: Demers et al. Nucl. Acids Res., (1995) 23, 3050-3055 and US Pat. No. 5,656,461). For this application, the
`unlabeled PNAs need to be designed such that they form PNA/nucleic add hybrids which are stable enough to disrupt
`the interstrand and intrastrand interactions of dsDNA. However, the PNA/nucleic acid complex must be susceptible to
`dissociation by the operation of the polymerase during primer extension.
`[0015]
`In still another related application, a process coined "PCR clamping" can be used to obtain point mutation
`discrimination when directing unlabeled PNAs of defined sequence to interfere with the PCR process (See: Ørum et
`al. Nucl. Acids Res. (1993), 21, 5332-5336). In one embodiment of PCR clamping, an unlabeled PNA, which is identical
`in nucleobase composition to the PCR primer, competes with the PCR primer for binding to the common recognition
`site. In another embodiment, the target site for the unlabeled PNA is located within the PCR amplicon region. In this
`embodiment, clamping operates if the PNA/nucleic acid hybrid is stable enough to prevent read through by the polymer-
`ase. In yet another embodiment, the target site for the unlabeled PNA is located adjacent to the PCR priming site. In
`this embodiment, PCR clamping may operate either by preventing read through of the polymerase or by preventing
`(blocking) primer annealing. To obtain point mutation discrimination using PCR clamping, longer mutant and wild type
`nucleic acid PCR primers are designed such that amplification proceeds only if the longer PCR primer is a perfect
`complement to the recognition site and thereby out competes the unlabeled PNA for binding within that site. PCR
`damping has recently been directed to analysis of the Ki-ras mutations of codon 12 and 13 (See: Thiede et al. Nucl.
`Acids Res. (1996) 24, 983-984).
`[0016] Very recently, the "Hybridization based screening on peptide nucleic acid (PNA) oligomer arrays" has been
`described wherein arrays of some 1000 PNA oligomers. of individual sequence were synthesized on polymer mem-
`branes (See: Weller et al. Nucl. Acids Res. (1997) 25, 2792-2799). Arrays are generally used, in a single assay, to
`generate affinity binding (hybridization) information about a specific sequence or sample to numerous probes of defined
`composition. Thus, PNA arrays may be useful in diagnostic or antisence applications. However, in the present study,
`the authors note that the affinity and spedficity of DNA hybridization to immobilized PNA oligomers depended on hy-
`bridization conditions more than was expected. Moreover, there was a tendency toward nonspecific binding at lower
`ionic strength. Furthermore, certain very strong binding mismatches were identified which could not be eliminated by
`more stringent washing conditions. These results demonstrate the need for improved methods of suppressing the
`binding of nucleic acids to non-complementary PNAs. Moreover, these unexplained results are also illustrative of the
`lack of complete understanding of these newly discovered molecules (i.e. PNA)
`[0017] There are indeed many differences between PNA probes and standard nucleic acid probes. These differences
`can be conveniently broken down into biological, structural, and physico-chemical differences. As discussed above
`and below, these biological, structural, and physico-chemical differences may lead to unpredictable results when at-
`tempting to use PNA probes in applications were nucleic acids have typically been employed. This non-equivalency
`of differing compositions is often observed in the chemical arts.
`[0018] With regard to biological differences, nucleic acids, are biological materials that play a central role in the life
`of living species as agents of genetic transmission and expression. Their in vivo properties are fairly well understood.
`PNA, on the other hand is recently developed totally artificial molecule, conceived in the minds of chemists and made
`using synthetic organic chemistry. It has no known biological function.
`[0019] Structurally, PNA also differs dramatically from nucleic acid. Although both can employ common nucleobases
`(A, C, G, T, and U), the backbones of these molecules are structurally diverse. The backbones of RNA and DNA are
`composed of repeating phosphodiester ribose and 2-deoxyribose units. In contrast, the backbones of PNA are com-
`posed on N-(2-aminoethyl)glycine units. Additionally, in PNA the nucleobases are connected to the backbone by an
`additional methylene carbonyl unit.
`[0020] Despite its name, PNA is not an acid and contains no charged acidic groups such as those present in DNA
`and RNA. Because they lack formal charge, PNAs are generally more hydrophobic than their equivalent nucleic acid
`molecules. The hydrophobic character of PNA allows for the possibility of non-specific (hydrophobic/hydrophobic in-
`teractions) interactions not observed with nucleic acids. Further, PNA is achiral, providing it with the capability of adopt-
`ing structural conformations the equivalent of which do not exist in the RNA/DNA realm.
`[0021] The physico/chemical differences between PNA and DNA or RNA are also substantial. PNA binds to its com-
`plementary nucleic acid more rapidly than nucleic add probes bind to the same target sequence. This behavior is
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`believed to be, at least partially, due to the fact that PNA lacks charge on its backbone. Additionally, recent publications
`demonstrate that the incorporation of positively charged groups into PNAs will improve the kinetics of hybridization
`(See: Iyer et al. J. BioL Chem. (1995) 270, 14712-14717). Because it lacks charge on the backbone, the stability of
`the PNA/nucleic acid complex is higher than that of an analogous DNA/DNA or RNA/DNA complex. In certain situations,
`PNA will form highly stable triple helical complexes or form small loops through a process called "strand displacement".
`No equivalent strand displacement processes or structures are known in the DNA/RNA world.
`[0022]
`In summary, because PNAs hybridize to nucleic acids with sequence speafiaty, PNAs are useful candidates
`for developing probe-based assays. However, PNA probes are not the equivalent of nucleic acid probes. Nonetheless,
`even under the most stringent conditions both the exact target sequence and a closely related sequence (e.g. a non-
`target sequence having a single point mutation (a.k.a. single base pair mismatch)) will often exhibit detectable inter-
`action with a labeled nucleic acid or labeled PNA probe (See: Nielsen et al. Anti-Cancer Drug Design at p. 56-57 and
`Weller et al. at p. 2798, second full paragraph). Any hybridization to a closely related non-target sequence will result
`in the generation of undesired background signal. Because the sequences are so closely related, point mutations are
`the some of the most difficult of all nucleic acid modifications to detect using a probe based assay. Numerous diseases,
`such as sickle cell anemia and cystic fibrosis, are caused by a single point mutation of genomic nucleic acid. Conse-
`quently, any method, kits or compositions which could improve the specificity, sensitivity and reliability of probe-based
`assays would be useful in the detection, analysis and quantitation of nucleic acid containing samples and particularly
`useful for nucleic add point mutation analysis.
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`Disclosure Of The Invention:
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`1. Summary:
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`[0023] This invention relates to methods, kits and compositions suitable for the improved detection, quantitation and
`analysis of nucleic acid target sequences using probe-based hybridization assays. The invention is more specifically
`directed to methods, kits and compositions suitable for suppressing the binding of detectable probes to non-target
`sequences in an assay for a target sequence of a nucleic acid target molecule. Suppression of the nonspecific binding
`of detectable probe directly improves the sensitivity of the assay thereby improving the signal to noise ratio of the
`assay. Suppression of nonspecific binding will also result in improvements in reliability since the incidence of false
`positives and false negative should also be reduced. Because the methods, kits and compositions of this invention are
`directed to the suppression of nonspecific binding of probes to nudeic acids, they are particularly well suited for the
`development of sensitive and reliable probe-based hybridization assays designed to analyze for point mutations. The
`methods, kits and compositions of this invention should also find utility for the detection, quantitation or analysis of
`organisms (micro-organisms), viruses, fungi and genetically based clinical conditions of interest.
`[0024]
`It has been surprisingly observed that the signal caused by the nonspecific binding of detectable probes to
`one or more non-target nudeic add sequences can be dramatically suppressed by the addition of one or more unlabeled
`probes wherein the sequence of the one or more unlabeled probes is complementary to one or more non-target se-
`quences to which the detectable probe binds in a nonspecific manner. For example, it has been observed that the
`addition of 25 equivalents of unlabeled PNA probe, having a single mismatch as compared with the labeled PNA probe,
`does not substantially alter the detection linut of the assay. However, the presence of the unlabeled PNA probe resulted
`in at least a 10 fold suppression in the binding of labeled PNA probe to the non-target sequence (point mutation) and
`a correlating improvement of approximately 30 fold, in the signal to noise ratio of the assay (see Example 4A and
`Figure 1).
`[0025] When the unlabeled PNA probe was present at 500 equivalents, there was very little loss of detectable signal
`(approximately 3 to 10 fold). However, suppression of binding of the labeled probe to a non-target sequence (point
`mutation) is substantially improved as compared with the experiment wherein only 25 equivalents of unlabeled PNA
`probe was present (Compare: Examples 4A and 4B of this specification). The results demonstrate that point mutation
`discrimination improved from approximately 10 fold in the absence of the unlabeled probe to greater than 1000 fold in
`the presence of high levels of unlabeled (blocker) PNA probe. Consequently, when employing the methods described
`herein, one can achieve several logs of improvement in point mutation discrimination and similar dramatic improve-
`ments in the dynamic range of the hybridization assay.
`[0026] The applicants are not aware of any similar method suitable for obtaining such a dramatic suppression of
`binding to non-target sequences and the correlating improvement in signal to noise ratio. The data presented in Ex-
`ample 6, demonstrates the clear superiority of PNA probes as compared with DNA probes with regard to suppression
`of binding to non-target sequences, improvement in signal to noise ratios and point mutation discrimination.
`[0027]
`In preferred embodiments of this invention, PNA probes are used either alone or in combination with nucleic
`acid probes. When combined with nucleic acid probes, the preferred combination involves unlabeled PNA probes used
`to suppress the binding of detectable (labeled). nucleic acid probes to non-target sequences. In the most preferred
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`embodiment of this invention, both the detectable probes and unlabeled or independently detectable probes are PNA
`probes because this embodiment exhibits both the greatest ability to suppress binding to non-target sequences and
`the greatest ability to discriminate point mutations.
`[0028] The hybridization assay of this invention can be performed in solution. Alternatively, one or more assay com-
`ponents may be immobilized to a surface. Thus, in one embodiment the nucleic acid target molecule comprising the
`target sequence is immobilized to a surface. In this embodiment, the immobilized target sequence is contacted with a
`solution containing the detectable and unlabeled or independently detectable probes (e.g. dot blot format). Alternatively,
`one or more probes may be immobilized on a surface and used, in a capture assay, to capture the nucleic acid target
`molecule comprising the target sequence. In a preferred embodiment, arrays of greater than two probes are used to
`generate binding (affinity) or sequence information about one or more nucleic acid target molecules of interest which
`may be present in the sample.
`[0029]
`In one embodiment, the invention is related to a method for suppressing the binding of detectable probe to a
`non-target sequence in an assay of a sample for a target sequence. The method comprises contacting the sample
`with a set containing two or more probes under conditions suitable for the probes to hybridize to nucleic acid. At least
`one of the probes is a detectable probe labeled with a detectable moiety and having a sequence complementary or
`substantially complementary to a target sequence. At least one of the other probes is an unlabeled or independently
`detectable probe having a sequence complementary or substantially complementary to a non-target sequence. The
`second step comprises detecting the presence, absence or quantity of a target sequence in the sample by directly or
`indirectly detecting or quantitating the detectable moiety. At least one of either a detectable probe or an unlabeled or
`independently detectable probe is a PNA probe. Preferably, the one or more unlabeled or independently detectable
`probes is a PNA probe. Most preferably, all the probes are PNA probes. In preferred embodiments, the detectable
`probe is perfectly complementary to a target sequence and the unlabeled or independently detectable probe is perfectly
`complementary to a non-target sequence which may be present in the sample.
`[0030]
`In another embodiment, the invention relates to a kit suitable for suppressing the binding of a detectable probe
`to a non-target sequence in an assay of a sample for a target sequence. The kit comprises a set of two or more probes
`wherein, at least one of the probes is a detectable probe labeled with a detectable moiety and having a sequence
`complementary or substantially complementary to the target sequence. At least one of the other probes is an unlabeled
`or independently detectable probe having a sequence complementary or substantially complementary to the non-target
`sequence. At least one of either a detectable probe or an unlabeled or independently detectable probe is a PNA probe.
`Preferably, the one or more unlabeled or independently detectable probes is a PNA probe. Most preferably, all the
`probes are PNA probes. In preferred embodiments, the detectable probe is perfectly complementary to a target se-
`quence and the unlabeled or independently detectable probe is perfectly complementary to a non-target sequence
`which may be present in the sample.
`[0031]
`In another embodiment, the invention relates to a composition for suppressing the binding of a detectable
`probe to a non-target sequence in an assay of a sample for a target sequence. The composition consists of a set of
`two probes wherein, one of the probes is a detectable probe labeled with a detectable moiety and having a sequence
`complementary to the target sequence. The other probe is an unlabeled or independently detectable probe having a
`sequence complementary to a non-target sequence. Either of the detectable probe or the unlabeled or independently
`detectable probe is a PNA probe. Preferably, the unlabeled or independently detectable probe is the PNA probe. Most
`preferably, both probes of the composition are PNA probes.
`[0032]
`In another embodiment, the invention relates to a composition for suppressing the binding of a detectable
`probe to a non-target sequence in an assay of a sample for a target sequence. The composition consists of a set of
`four probes wherein, one of the probes is a detectable probe labeled with a detectable moiety and having a sequence
`complementary to the target sequence. The other three probes are unlabeled or independently detectable probes
`having sequences which hybridize specifically with non-target sequences which are related to the target sequence as
`the three possible single point mutations. Either of the detectable probe or at least one of the three unlabeled or
`independently detectable probes is a PNA probe. Preferably the three unlabeled or independently detectable probes
`are PNA probes. Most preferably, all probes of the composition are PNA probes.
`[0033]
`In yet another embodiment, this invention relates to a method for suppressing the binding of a non-target
`sequence to a capture probe immobilized on a surface in a capture assay of a sample for a target sequence. The
`method comprises contacting the sample with a solution containing on