# Patent

## Title
Suppression of non-specific amplification with high-homology oligonucleotides

## Abstract

The invention comprises suppressor oligonucleotides for reducing amplification of a non-target nucleic acid sequences; the method of designing and using such oligonucleotides, as well as kits and reaction mixtures.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 20, 2012, is named 30622US1.txt and is 27 bytes in size.
BACKGROUND OF THE INVENTION
Amplification of nucleic acids by polymerase chain reaction (PCR) has many applications in biomedical research, diagnostics and biotechnology. The unique specificity of PCR enables selective amplification of a particular nucleic acid sequence in the presence of overwhelming amount of other sequences. Furthermore, PCR can distinguish a target sequence from another sequence that is different by as little as a single base-pair. For example, allele-specific PCR (AS-PCR) is capable of detecting small alterations in DNA and even single nucleotide mutations in the presence of the wild-type, non-mutant DNA (U.S. Pat. No. 6,627,402). In an allele-specific PCR assay, at least one primer is allele-specific, i.e. designed to preferentially match the target sequence (a specific variant of the sequence), but contains discriminating mismatches with non-target sequences (other variants of the sequence). Ideally, primer extension occurs only when the allele-specific primer is hybridized to the target sequence. In a successful allele-specific PCR, the target variant of the nucleic acid is amplified, while the other non-target variants are not, at least not to a detectable level. Unfortunately, with many targets, this ideal is not achievable. It is common that in later cycles of PCR, amplification of the non-target variants of the sequence also becomes detectable. This phenomenon is called “breakthrough amplification.” Even though the AS-PCR primers are perfectly complementary (or at least, share the greater degree of complementarity) with the target sequence and are mismatched (or have more mismatches) with non-target sequences, often amplification of the non-target sequences cannot be completely avoided.
Breakthrough amplification is of special concern in assays where the sample contains small amounts of the target sequence and large amounts of the non-target sequence. For example, in an assay targeting a somatic mutation in a tumor, only a fraction of cells from the patient's sample are tumor cells. A fraction of tumor cells may contain mutations indicating susceptibility to a particular anti-tumor drug (mutations described in U.S. Pat. Nos. 7,294,468 and 7,960,118). In such a sample, a small number of the target (mutant) sequences are mixed with a large number of non-target (non-mutant) sequences. Breakthrough amplification of the non-mutant sequence would produce a false-positive result, falsely indicating the presence of a mutation and misdirecting the patient's therapy. If the specificity of the assay is limited by the breakthrough amplification, so is the clinical utility of the assay.
Various means of preventing or reducing non-specific amplification have been proposed (for example, chemical modifications that affect the specificity of amplification primers, see U.S. Pat. No. 6,011,611; using a blocker oligonucleotide, see U.S. Application Pub. No. 200953720). However, these methods are not always successful in entirely eliminating the breakthrough amplification. Accordingly, there is a need for alternative methods of preventing or minimizing breakthrough amplification in a nucleic acid amplification reaction.
SUMMARY OF THE INVENTION
In one embodiment, the invention is a suppressor oligonucleotide for use in a nucleic acid amplification reaction, having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism.
In another embodiment, the invention is a method of designing a suppressor oligonucleotide for use in a nucleic acid amplification reaction, comprising using sequence alignment algorithms to select an oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism.
In yet another embodiment, the invention is a method of reducing amplification of a non-target nucleic acid template in a nucleic acid amplification reaction, comprising performing the amplification reaction in the presence of a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism.
In yet another embodiment, the invention is a kit for performing an amplification reaction with reduced amplification of the non-target sequences, comprising a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism.
In yet another embodiment, the invention is a reaction mixture for performing an amplification reaction with reduced amplification of the non-target sequences, comprising a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism.
In yet another embodiment, the invention is the use of a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism, in a nucleic acid amplification reaction to reduce non-specific amplification.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows amplification of exon 2 (including codon 12) of the human NRAS gene by allele-specific PCR with breakthrough suppression by the suppressing oligonucleotide, also used as one of the primers. FIG. 1A shows unsuppressed breakthrough amplification (dashed line), and FIG. 1B shows suppression of the non-target sequence amplification.
FIG. 2 shows amplification of exon 3 (including codon 61) of the human NRAS gene by allele-specific PCR with breakthrough suppression by the suppressing oligonucleotide that is not complementary to the target sequence. FIG. 2A shows no suppression of the breakthrough amplification without the suppressor oligonucleotide, FIG. 2B shows no suppression when the suppressor oligonucleotide was present at low concentration and FIG. 2C shows suppression when the suppressor oligonucleotide was present at a higher relative concentration.
FIG. 3 shows amplification of the human PI3KCA gene by allele-specific PCR with breakthrough suppression by the suppressing oligonucleotide that is not complementary to the target sequence. FIG. 3A shows breakthrough amplification in the absence of the suppressor oligonucleotide and FIG. 3B shows suppression of the breakthrough amplification in the presence of the suppressor oligonucleotide.
FIG. 4 shows amplification of the human BRAF gene (including codons 469 and 600) by allele-specific PCR with breakthrough amplification suppression by the suppressing oligonucleotide that is not complementary to the target sequence. FIG. 4A shows breakthrough amplification in the absence of the suppressor oligonucleotide and FIG. 4B shows suppression of the breakthrough amplification in the presence of the suppressor oligonucleotide in the codon 469 reaction. FIG. 4C shows breakthrough amplification in the absence of the suppressor oligonucleotide and FIG. 4D shows suppression of the breakthrough amplification in the presence of the suppressor oligonucleotide in the codon 600 reaction.
FIG. 5 shows amplification of exons 2 and 3 of the human NRAS gene by allele-specific PCR with breakthrough suppression by simultaneous linear amplification of the M13 target. FIG. 5A shows breakthrough amplification of the non-target (wild-type) NRAS sequence in the presence of a primer pair consisting of an allele-specific primer matched to one of the mutations in codon 61 and a common primer. FIG. 5B shows suppression of breakthrough amplification of the non-target (wild-type) NRAS sequence by M13 DNA and three primers capable of linear amplification of the M13 DNA. FIG. 5C shows breakthrough amplification of the non-target (wild-type) NRAS sequence in the presence of an allele-specific primer matched to one of the mutations in codon 12 and a common primer. FIG. 5D shows suppression of breakthrough amplification of the non-target (wild-type) NRAS sequence M13 DNA and three primers capable of linear amplification of the M13 DNA.
FIG. 6 shows amplification of exon 2 of the human NRAS gene by allele-specific PCR with breakthrough suppression by suppressing oligonucleotides with varying degrees of homology to the target genome. FIG. 6A shows no suppression of the breakthrough amplification by a suppressor oligonucleotide with low degree of homology; FIG. 6B shows partial suppression by an oligonucleotide with medium degree of homology; and FIG. 6C shows complete suppression by an oligonucleotide with high degree of homology.
FIG. 7 shows results of a BLAST® search for the regions of interest in exon 2 of human NRAS gene for the design of suppressing oligonucleotides.
FIG. 8 shows an example of selecting suppressing oligonucleotides from the region of interest in exon 2 of human NRAS gene.



DETAILED DESCRIPTION OF THE INVENTION
To facilitate the understanding of this disclosure, the following definitions of the terms used herein are provided.
The term “allele-specific primer” or “AS primer” refers to a primer that may hybridize to more than one variant of the target sequence, but is capable of discriminating among variants of the target sequence, such that efficient extension of the primer by the nucleic acid polymerase under suitable conditions occurs only upon hybridization of the primer to one particular variant. With other variants of the target sequence, the extension is less efficient or inefficient.
The term “amplicon” refers to a nucleic acid formed as a product of a polymerase chain reaction.
The term “common primer” refers to the second primer in the pair of primers that includes an allele-specific primer. The common primer is not allele-specific, i.e. does not discriminate between the variants of the target sequence between which the allele-specific primer discriminates.
The terms “complementary” or “complementarity” are used in reference to antiparallel strands of polynucleotides related by the Watson-Crick base-pairing rules. Complementary nucleic acid strands are capable of forming duplexes under standard hybridization conditions. The terms “perfectly complementary” or “100% complementary” refer to complementary sequences that have Watson-Crick pairing of all the bases between the antiparallel strands, i.e. there are no mismatches between any two bases in the polynucleotide duplex. The terms “partially complementary” or “incompletely complementary” refer to any alignment of bases between antiparallel polynucleotide strands that is less than 100% perfect (e.g., there exists at least one mismatch or unmatched base in the polynucleotide duplex). A smaller nucleic acid strand (e.g. an oligonucleotide) may be complementary to a region (site) in a larger nucleic acid, e.g. a gene or a genome. Under standard hybridization conditions, duplexes are formed between antiparallel strands even in the absence of perfect complementarity. However, duplexes between partially complementary strands are generally less stable than the duplexes between perfectly complementary strands.
A “growth curve” in the context of a nucleic acid amplification assay is a graph of a function, where an independent variable is the number of amplification cycles and a dependent variable is an amplification-dependent measurable parameter measured at each cycle of amplification. Typically, the amplification-dependent measurable parameter is the amount of fluorescence emitted by the probe upon hybridization, or upon the hydrolysis of the probe by the nuclease activity of the nucleic acid polymerase, see Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Pat. No. 5,210,015. In a typical polymerase chain reaction, a growth curve comprises a segment of exponential growth followed by a plateau. A growth curve is typically characterized by a “cycles to threshold” value or “Ct” value, which is a number of cycles where a predetermined magnitude of the measurable parameter is achieved. A lower or “earlier” Ct value represents more rapid amplification, while the higher or “later” Ct value represents slower amplification.
The terms “homology” and “regions of homology” refer to regions (sites) where two nucleic acids share at least partial complementarity. A region of homology may span only a portion of the sequences. For example, only a portion of an oligonucleotide may be homologous to a site in the genome. Different portions of the oligonucleotide may be homologous to several distinct sites in the genome, while an entire oligonucleotide may be homologous to yet another site in the genome. As with any partially complementary nucleic acid sequences, a region of homology may contain one or more mismatches and gaps when the two sequences are aligned. A smaller nucleic acid strand (e.g. an oligonucleotide) may be homologous to a region (site) in a larger nucleic acid, e.g. a gene or a genome. The term “degree of homology” between two sequences refers to the extent of identity between the sequences. The extent of identity is commonly expressed as a ratio of mismatched nucleotides in the homologous region to the total number of nucleotides, expressed in percentage. For example, a 20-base oligonucleotide that hybridizes to a homologous region (site) in the target genome with two mismatches is said to have 90% identity to that region. The term “degree of homology to the target genome” is a measure of the number and percent identity of regions of homology to the oligonucleotide present in the target genome. An oligonucleotide with high degree of homology has many regions of homology with high percentage of identity throughout the target genome, while an oligonucleotide with low degree of homology region would have fewer regions of homology with low percentage of identity in the target genome.
The terms “hybridized” and “hybridization” refer to the base-pairing interaction between two at least partially complementary (as defined herein) nucleic acid strands which results in formation of a duplex. It is not a requirement that two nucleic acids have 100% complementarity over their full length to achieve hybridization. A smaller nucleic acid strand (e.g. an oligonucleotide) may hybridize to a region (site) in a larger nucleic acid, e.g. a gene or a genome.
The term “multiple regions of homology” in relation to suppressor oligonucleotides homologous to regions of a target genome is used to describe the number of such regions in the target genome that is sufficient to support the suppressing property of the oligonucleotide. In general, “multiple” means more than one, for example, 2, 3, 20, 30, 200, 300, 2000, 3000, etc., and any whole number in between. However, a sufficient number varies depending on the complexity of the target genome, i.e. for less complex genomes, a smaller number may be sufficient for the suppression phenomenon to occur, while for more complex genomes, a greater number would be required.
The terms “nucleic acid,” “oligonucleotide” and “polynucleotide” are used interchangeably to describe polymers of deoxyribo- (or ribo-) nucleic acid, including primers, probes, genomic DNA or RNA of various organisms and fragments of genomic DNA or RNA as well as other genetic elements, e.g. plasmids, cosmids, etc. The terms are not limited by length and are generic to polymers of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. These terms include double- and single-stranded nucleic acids. Nucleic acids can comprise naturally occurring phosphodiester linkages or modified linkages including, but not limited to thioesther linkages. Likewise, nucleic acids can comprise the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil) or other modified, non-standard, or derivatized base moieties.
The terms “polynucleotide” and “oligonucleotide” are used interchangeably. “Oligonucleotide” is a term sometimes used to describe a shorter polynucleotide. An oligonucleotide may be comprised of at least 6 nucleotides and up to 100 nucleotides.
The term “primary sequence” refers to the sequence of nucleotides in a polynucleotide or oligonucleotide. Nucleotide modifications such as nitrogenous base modifications, sugar modifications or other backbone modifications are not a part of the primary sequence. Labels, such as chromophores conjugated to the oligonucleotides are also not a part of the primary sequence. Thus two oligonucleotides can share the same primary sequence but differ with respect to modifications and labels.
The term “primer” refers to an oligonucleotide which hybridizes with a sequence in the target nucleic acid and is capable of acting as a point of initiation of synthesis along a complementary strand of nucleic acid under conditions suitable for such synthesis. A perfect complementarity is not required for the primer extension to occur. However, a primer with perfect complementarity (especially near the 3′-terminus) will be extended more efficiently than a primer with mismatches, especially mismatches at or near the 3′-terminus.
The term “probe” refers to an oligonucleotide which hybridizes with a sequence in the target nucleic acid and may be detectably labeled. The probe can have modifications, such as a 3′-terminus modification that makes the probe non-extendable by nucleic acid polymerases; and one or more chromophores. An oligonucleotide with the same sequence may serve as a primer in one assay and a probe in a different assay.
The term “region of interest” refers to a region of the target genome from which the suppressor oligonucleotide is to be designed.
The term “sample” refers to any composition containing or presumed to contain nucleic acid. This includes a sample of tissue or fluid isolated from an individual. For example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs and tumors, and also samples of in vitro cultures established from cells taken from an individual, including the formalin-fixed paraffin embedded tissues (FFPET) and nucleic acids isolated therefrom.
The term “suppressor oligonucleotide” refers to an oligonucleotide that, when present in the PCR mixture, suppresses or detectably reduces amplification of any non-target sequences. In some instances, the suppressor oligonucleotide detectably reduces exponential amplification of the non-target sequence in allele-specific PCR. The suppressor oligonucleotide may optionally, have additional functions, including serving as a primer for amplification of the target sequence.
A “template” or “target” refers to a nucleic acid which is to be amplified, detected or both. The target or template is a sequence to which a primer or a probe can hybridize. Template nucleic acids can be derived from essentially any source, including microorganisms, complex biological mixtures, tissues, bodily fluids, sera, preserved biological samples, environmental isolates, in vitro preparations or the like. The template or target may constitute all or a portion of a nucleic acid molecule.
The term “target organism” refers to an organism whose nucleic acid sample is being analyzed. The genome of the target organism is referred to as “target genome.”
The term “target sequence” refers to the sequence of the target organism of which amplification is desired. The term “non-target sequence” refers to another sequence of which amplification is not desired and is to be avoided. In the context of allele-specific PCR, the non-target sequence of concern is often a very similar variant of the target sequence. Although it is not desired, the non-target sequence is sometimes amplified by allele-specific PCR along with the target sequence, but with lower efficiency.
Polymerase chain reaction (PCR) is capable of specifically amplifying a target nucleic acid sequence present amidst a much larger number of other sequences. Allele-specific PCR (AS-PCR) is a method capable of distinguishing between sequences that differ by as little as a single nucleotide. The sensitivity and specificity of PCR and AS-PCR is such that the target variant of the nucleic acid can be selectively amplified even in the presence of much larger amounts of non-target variants and unrelated sequences. Ideally, the non-target nucleic acids are never amplified to a detectable level. However, sensitivity of PCR and AS-PCR assays is challenged by a phenomenon called “breakthrough amplification,” which is detectable amplification of the non-target nucleic acid sequences during the later cycles of PCR.
In conducting allele-specific PCR, the inventors discovered that certain oligonucleotides (initially used as primers) significantly reduce breakthrough amplification when present in AS-PCR assays (Example 1, FIG. 1). When these suppressor oligonucleotides were further investigated, it was discovered that most surprisingly, the oligonucleotides exert the same effect on unrelated targets, i.e. targets that have no regions of complementarity with the suppressor oligonucleotides, (Example 2, FIG. 2, Example 3, FIG. 3, and Example 4, FIG. 4). Accordingly, the inventors devised methods of designing and using such oligonucleotides for improving PCR and AS-PCR assays.
While not wishing to be bound by a particular theory, the inventors hypothesize that one of the mechanisms of breakthrough suppression may be sequestering PCR reagents in the later cycles of amplification when the breakthrough amplification usually occurs. In the later cycles of PCR, amplification of the target sequence ceases (the plateau is reached), in part because re-annealing of double-stranded amplicons is kinetically favored over annealing of primers to single strands of denatured amplicons. At that stage, excess primers become available for the less specific (and thus less efficient) breakthrough amplification that involves extension of a mismatched primer hybridized to the non-target sequence. However, thermodynamic parameters of the mismatched primer extension are unfavorable. Accordingly, the mismatched primer extension is greatly affected by the depletion or sequestering of components such as nucleotides and nucleic acid polymerase. The properties of the suppressor oligonucleotide allow for linear primer extension elsewhere in the genome and (optionally) for exponential generation of additional amplicons elsewhere in the genome. These extraneous reactions, although arguably not very efficient themselves, sequester critical reagents and inhibit breakthrough amplification requiring these reagents.
To test this hypothesis, the inventors conducted an experiment described in Example 5. In that example, an AS-PCR assay known for its breakthrough amplification (FIG. 5A) was conducted in the presence of an engineered primer/target combination capable of priming multiple linear extension reactions. The multiple linear extension reactions were predicted to generate some of the depletion effect and suppress the breakthrough amplification. Indeed, some suppression of the breakthrough amplification was observed (FIG. 5B).
In one embodiment, the invention is a suppressor oligonucleotide for suppressing amplification of non-target sequences in an amplification reaction, for example, PCR or allele-specific PCR (AS-PCR). The suppressor oligonucleotide is homologous to multiple sites in the genome of the target organism. These sites in the target genome comprise regions of homology with the suppressor oligonucleotide. In some embodiments, the regions of homology between the suppressor oligonucleotide and the target genome have at least 75% identity. In some embodiments, the regions of homology are at least 15 base pairs long. However, it is understood that for certain sequences (for example, GC-rich sequences) shorter regions of homology or regions with less than 75% identity may also offer satisfactory results. Generally, the higher the identity in each of the regions of homology, the better the suppressing effect as demonstrated in Example 6, FIG. 6. In yet other embodiments, the region of homology spans the 3′-end of the suppressor oligonucleotide. In yet other embodiments, within the last four base pairs at the 3′-end of the oligonucleotide, the region of homology contains no more than 2 mismatches.
It is desirable that the suppressor oligonucleotide cause minimal interference with amplification and detection of the target sequence. If a suppressor oligonucleotide is capable of generating additional (non-target) amplicons, these additional amplicons may be detected, and thus interfere with detection of the target sequence. Generation of these amplicons by the suppressor oligonucleotide is preferably avoided. In variations of this embodiment, the suppressor oligonucleotide possesses an additional property: it is not capable of generating additional amplicons. A PCR amplicon is generated in an exponential fashion only when both forward and reverse primers are present. Therefore an oligonucleotide is capable of priming exponential synthesis of an amplicon if it is paired with another oligonucleotide (including itself) that is capable of hybridizing to a sequence on the opposite strand of the same nucleic acid, said sequence located no more than approximately 1000 base pairs away from the site of the hybridization of the first oligonucleotide. It is understood that in some instances, for example when a highly processive nucleic acid polymerase is used (see e.g. U.S. Pat. No. 7,855,055), non-target amplicons longer than 1000 base pairs may also be generated and interfere with amplification and detection of the target nucleic acid. Accordingly, when a highly processive polymerase is used, a potential suppressor oligonucleotide may be excluded based on an upper limit higher than 1000 base pairs. In that case, more potential suppressor oligonucleotides would be excluded. On the other hand, with fragmented nucleic acid (for example, nucleic acid isolated from formalin-fixed paraffin-embedded tissues, FFPET), longer amplicons are not possible and a potential suppressor oligonucleotide may be excluded based on a limit shorter than 1000 base pairs. In that case, fewer potential suppressor oligonucleotides would be excluded. According to the present invention, in some embodiments, an oligonucleotide is not used as a suppressor oligonucleotide if it has at least two regions of homology located on the opposite strands of the target genome, said regions having at least 75% identity between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs.
A suppressor oligonucleotide can be prepared by any suitable method of preparing an oligonucleotide, usually chemical synthesis using commercially available reagents and instruments. Alternatively, an oligonucleotide can be purchased through commercial sources. Methods of synthesizing oligonucleotides are well known in the art (see, Narang et al., Meth. Enzymol. 68:90-99, 1979; Brown et al., Meth. Enzymol. 68:109-151, 1979; Beaucage et al., Tetrahedron Lett. 22:1859-1862, 1981; or U.S. Pat. No. 4,458,066).
In variations of this embodiment, the invention comprises suppressor oligonucleotides of SEQ ID NOs: 1-5 (Table 1).
In another embodiment, the invention is a method of designing a suppressor oligonucleotide for suppressing amplification of non-target sequences in amplification reaction, for example, PCR or allele-specific PCR (AS-PCR). The method of designing suppressor oligonucleotides of the present invention relies on sequence alignment algorithms. In some embodiments, oligonucleotide design method of the present invention uses sequence alignment software. Such software is currently widely available and in many instances, is accessible to the public free of charge. For example, National Institutes of Health has made available free of charge through its website the BLAST® (Basic Local Alignment Search Tool) software package. The invention is not limited to the use of BLAST®, but rather BLAST® is merely an example of a suitable software package. Other examples of pairwise sequence alignment software include ACANA (Huang et al. (2006) Accurate anchoring alignment of divergent sequences. Bioinformatics 22:29-34), Bioconductor (open-source software freely distributed by the Fred Hutchinson Cancer Research Center), FEAST (software package distributed free of charge by the University of Waterloo, Canada), FASTA (software package distributed free of charge by the University of Virginia), REPuter (Kurtz et al. (2001) REPuter: The Manifold Applications of Repeat Analysis on a Genomic Scale, Nucleic Acids Res., 29(22):4633-4642), SWIFT BALSAM (BAsic fiLter for Semigobal non-gapped AlignMent search) (Rasmussen et al. (2006) Efficient q-Gram Filters for Finding All epsilon-Matches over a Given Length, J. Comp. Biol. 13(2), 296-308).
In one embodiment, the method of the present invention comprises the use of sequence alignment algorithms to select an oligonucleotide characterized by having multiple regions of homology with the target genome. In some embodiments, the method uses sequence alignment algorithms to select an oligonucleotide where the regions of homology between the suppressor and the target genome have at least 75% identity. In some embodiments, the method uses sequence alignment algorithms to select an oligonucleotide where the regions of homology are at least 15 base pairs long. In yet other embodiments, the method uses sequence alignment algorithms to select an oligonucleotide where the regions of homology span the 3′-end of the oligonucleotide. In yet other embodiments, the method uses sequence alignment algorithms to select an oligonucleotide where within the last four base pairs at the 3′-end of the oligonucleotide, the region of homology contains no more than 2 mismatches.
In variations of this embodiment, the method of the present invention comprises the use of sequence alignment algorithms to exclude an oligonucleotide from use as a suppressor oligonucleotide if the oligonucleotide has at least two regions of homology located on the opposite strands of the target genome, said regions having at least 75% identity between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs.
In some embodiments of the invention, the suppressor oligonucleotide is derived from a region of interest selected by the user. The region of interest may contain or be adjacent to the target sequence, or may be an unrelated region of the genome. There is no limitation on the size of the region of interest, although generally a larger region may yield more options for the design of the suppressor oligonucleotides. In general, the region of interest should possess some of the characteristics desired in the suppressor oligonucleotides. In some embodiments of the method, the region of interest comprises multiple regions of homology with the target genome that have at least 75% identity and are at least 15 nucleotides long.
In one embodiment, the method of the present invention comprises the following steps performed with the use of sequence alignment algorithms:
(a) identify one or more regions of interest;
(b) conduct a search of the target genome sequence using the regions of interest as a query to identify regions of homology between the region of interest and the target genome;
(c) select sections of the region of interest having the most regions of homology to the target genome;
(d) design one or more oligonucleotides in the sections selected in step (c);
(e) conduct a search of the target genome with the oligonucleotides designed in step (d) to identify the oligonucleotides with the maximum number of regions of homology to the target genome meeting one or both of the following criteria: at least 75% identity and no more than 2 mismatches present in the 3′-terminal region of the oligonucleotide;
(f) optionally, conduct a search of the target genome with the oligonucleotides designed in step (d) to identify and exclude the oligonucleotides having at least two regions of homology located on the opposing strands of the target genome sequence that are separated by fewer than approximately 1000 base pairs.
In general, the region of interest and the oligonucleotide with the most regions of homology identified in step (e), and optionally, selected as not capable of generating a non-target amplicon (f) are to be selected. It is however understood, that an excessive number of regions of homology may be detrimental to the assay as a whole. For example, an oligonucleotide homologous to a highly repetitive element in the target genome will initiate an excessive number of primer extensions that will overwhelm the reaction. See e.g. Kazazian, H (2004) Mobile Elements: Drivers of Genome Evolution, Science 303 (5664): 1626-1632 (Alu repetitive element constitutes 11% of the human genome, i.e. occurs about 3×108 times throughout the genome).
Example 6 demonstrates application of the method. FIG. 7 is an illustration of steps (a) through (c) performed using BLAST®. FIG. 8 is an illustration of steps (d) through (e) performed using BLAST®.







TABLE 1







Suppressor oligonucleotides










SEQ ID NO:
Sequence 5′-3′







SEQ ID NO: 1
CTACCACTGGGCCTCACCT



 



SEQ ID NO: 2
CAGGATCAGGTCAGCGGGCT



 



SEQ ID NO: 3
AGACAGGATCAGGTCAGCGGG



 



SEQ ID NO: 4
CAGGTCAGCGGGCTACCACT



 



SEQ ID NO: 5
ACAAGTGAGAGACAGGATCAGGTC










For successful extension of a primer, the primer needs to have at least partial complementarity to the target sequence. Generally, complementarity at the 3′-end of the primer is more critical than complementarity at the 5′-end of the primer. (Innis et al. Eds. PCR Protocols, (1990) Academic Press, Chapter 1, pp. 9-11). Therefore the present invention encompasses the oligonucleotides disclosed in Table 1, as well as variants of these oligonucleotides with 5′-end variations.
In one embodiment, the invention is a method of suppressing amplification of a non-target sequence in an amplification reaction, for example, PCR or allele-specific PCR (AS-PCR), comprising conducting the AS-PCR in the presence of a suppressor oligonucleotide that is homologous to multiple sites in the genome sequence of the target organism. In some embodiments, the regions of homology between the suppressor oligonucleotide and the target genome have at least 75% identity. In some embodiments, the regions of homology are at least 15 base pairs long. In yet other embodiments, the region of homology spans the 3′-end of the suppressor oligonucleotide. In yet other embodiments, within the last four base pairs of the 3′-end of the oligonucleotide, the region of homology contains no more than 2 mismatches. In yet other embodiments, an oligonucleotide is not used as a suppressor oligonucleotide if it has at least two regions of homology located on the opposite strands of the target genome, said regions of homology having at least 75% identity between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs.
The method of the present invention is applicable to traditional PCR as well as allele-specific PCR. Allele-specific PCR is a variation of PCR where the primers are designed to amplify the target sequence but avoid amplification of another, closely related sequence. Allele-specific PCR is described e.g. in U.S. Pat. No. 6,627,402. In allele-specific PCR, at least one of the primers is the discriminating primer having a sequence complementary to the target sequence, but having mismatches with the non-target sequence. Typically, the discriminating nucleotide in the primer, i.e. the nucleotide matching only the target sequence, is the 3′-terminal nucleotide. In cases where the primer is not perfectly complementary to the target sequence, it still comprises a greater degree of complementarity to the target sequence compared to the non-target sequence. Design of allele-specific primers and general methods of optimizing the primers for nucleic acid amplification have been described, for example, in PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., (1990) Academic Press.
Typically, primers are synthetic oligonucleotides, composed of A, C, G and T nucleotides. However, unconventional base nucleotides, not normally found in nucleic acids, can also be used. For example, certain modified bases are known to increase specificity of amplification, see U.S. Pat. No. 6,001,011. Innis et al. (supra) also contains guidance on selecting nucleic acid polymerases for use in PCR. Exemplary thermostable DNA polymerases include those from Thermus thermophilus, Thermus caldophilus, Thermus sp. ZO5 (see, e.g., U.S. Pat. No. 5,674,738), Thermus aquaticus, Thermus flavus, Thermus filiformis, Thermus sp. sps17, Deinococcus radiodurans, Hot Spring family B/clone 7, Bacillus stearothermophilus, Bacillus caldotenax, Thermotoga maritima, Thermotoga neapolitana and Thermosipho africanus. 
Detection of the amplification products may be accomplished by any method known in the art. These detection methods include the use of labeled primers and probes as well as various nucleic acid-binding dyes. The means of detection may be specific to one variant of the target sequence, or may be generic to all variants of the target sequence or even to all double stranded DNA. The amplification products may be detected after the amplification has been completed, for example, by gel electrophoresis of the unlabeled products and staining of the gel with a nucleic acid-binding dye. Alternatively, the amplification products may carry a radioactive or a chemical label, either by virtue of incorporation during synthesis or by virtue of having a labeled primer. After or during electrophoresis, the labeled amplification products may be detected with suitable radiological or chemical tools known in the art. After electrophoresis, the product may also be detected with a target-specific probe labeled by any one of the methods known in the art. The labeled probe may also be applied to the target without electrophoresis, i.e. in a “dot blot” assay or the like.
In some embodiments, the presence of the amplification product may be detected in a homogeneous assay, i.e. an assay where the nascent product is detected during the cycles of amplification, and no post-amplification handling is required. A homogeneous amplification assay using a nuclease probe has been described for example, in U.S. Pat. No. 5,210,015. Homogeneous amplification assay using nucleic acid-intercalating dyes has been described for example, in U.S. Pat. Nos. 5,871,908 and 6,569,627. The homogeneous assay may also employ one or more fluorescent probes where hybridization of the probes to the extension product results in enzymatic digestion of the probe and detection of the resulting fluorescence (TaqManTM probe method, Holland et al. (1991) P.N.A.S. USA 88:7276-7280). Other methods use two probes labeled with two interacting fluorophores. The examples of such probes include “molecular beacon” probes (Tyagi et al., (1996) Nat. Biotechnol., 14:303-308) or fluorescently labeled nuclease probes (Livak et al., (1995) PCR Meth. Appl., 4:357-362).
In a homogeneous assay, the reaction is characterized by a growth curve showing the increase in fluorescence of a probe with each cycle of PCR. See Holland et al., (supra) and U.S. Pat. No. 5,210,015. Each growth curve is characterized by a “cycles to threshold” value or “Ct” value. A lower Ct value represents more rapid completion of amplification, while the higher Ct value represents slower completion of amplification. A lower Ct value may also represent a greater initial input of the target nucleic acid, while a higher Ct value may represent a smaller initial input. In the case of allele-specific PCR however, the lower Ct value represents efficient amplification. During breakthrough amplification, the non-target sequence yields a very high Ct value despite the large amount of the non-target sequence present. The high Ct value reflects very inefficient amplification of the non-target nucleic acid.
In yet another embodiment, the invention is a kit containing reagents necessary for performing an amplification reaction, for example PCR or AS-PCR, with reduced amplification of non-target sequences. The reagents comprise one or more allele-specific primers, one or more corresponding common primers and optionally, one or more probes; and a suppressor oligonucleotide characterized by having multiple regions of homology with the target genome. In some embodiments, the regions of homology have one or more of the following properties: at least 75% identity between the suppressor oligonucleotide and the target genome sequence; at least 15 base pairs long; span the 3′-end of the suppressor oligonucleotide; and within the last four base pairs at the 3′-end of the oligonucleotide, the regions of homology contains no more than 2 mismatches. In yet other embodiments, an oligonucleotide is not included in the kit as a suppressor oligonucleotide if it has at least two regions of homology located on the opposite strands of the target genome, said regions of homology having at least 75% identity between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs.
The kit may further comprise reagents necessary for the performance of an amplification and detection assay, such as nucleoside triphosphates, nucleic acid polymerase and buffers necessary for the function of the polymerase. In some embodiments, the probe is detectably labeled. In such embodiments, the kit may comprise reagents for detecting the label. Optionally, the kit may also contain reagents that enhance the performance of the PCR, including dUTP and uracil-N-glycosylase (UNG) to reduce contamination, and betaine to improve specificity.
In yet another embodiment, the invention is a reaction mixture for performing an amplification reaction, for example, PCR or allele-specific PCR, with reduced amplification of non-target sequences. The mixture comprises one or more allele-specific primers, one or more corresponding common primers and optionally, one or more probes; and a suppressor oligonucleotide characterized by having multiple regions of homology with the target genome. In some embodiments, the regions of homology have one or more of the following properties: at least 75% identity between the suppressor oligonucleotide and the target genome sequence; at least 15 base pairs long; span the 3′-end of the suppressor oligonucleotide; and within the last four base pairs of the 3′-end of the oligonucleotide, the region of homology contains no more than 2 mismatches. In yet other embodiments, an oligonucleotide is not included in the reaction mixture as a suppressor oligonucleotide if it has at least two regions of homology located on the opposite strands of the target genome, said regions of homology having at least 75% identity between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs. The reaction mixture may further comprise reagents such as nucleoside triphosphates, nucleic acid polymerase and buffers necessary for the function of the polymerase.
EXAMPLES
Example 1
Suppression of Breakthrough Amplification by a PCR Primer
In this example, suppression of breakthrough amplification was observed in an AS-PCR targeting mutations in codon 12 of the human NRAS gene. The primers and probes used in Example 1 are shown in Table 2. An upstream primer selected from among SEQ ID NOs: 6-23 is matched to one of the mutations 35G>C, 34G>T, 35G>A, 34G>C, 34G>A, and 35G>T corresponding to amino acid changes G12A, G12C, G12D, G12R, G12S, and G12V in exon 2 of the human NRAS gene and is mismatched with the wild-type sequence. A downstream primer selected from SEQ ID NOs: 24-26 is common between the mutant and wild-type sequences of exon 2 in the human NRAS gene and the detection probe is selected from SEQ ID NOs: 27-29.







TABLE 2







Primers and probes for exon 2 of the NRAS gene used in Example 1.









SEQ ID NO:
Function
Sequence 5′-3′





SEQ ID NO: 6
35G > C AS primer
CTGGTGGTGGTTGGAGCCGC


 


SEQ ID NO: 7
35G > C AS primer
CTGCTGGTGGTTGGAGEAGC


 


SEQ ID NO: 8
35G > C AS primer
CTGCTGGTGGTTGGAGCMGC


 


SEQ ID NO: 9
34G > T AS primer
CAAACTGGTGGTGGTTGGAGCTT


 


SEQ ID NO: 10
34G > T AS primer
TACAAACTGGTGGTGGTTGGAGCTT


 


SEQ ID NO: 11
34G > T AS primer
CAGAGTGGTGGTGGTTGGAGCDT


 


SEQ ID NO: 12
35G > A AS primer
AAGTGGTGGTGGTTGGAGCDGA


 


SEQ ID NO: 13
35G > A AS primer
AACTTGGTGGTGGTTGGAGTMGA


 


SEQ ID NO: 14
35G > A AS primer
AACTGGTGGTGGTTGGAGCTGA


 


SEQ ID NO: 15
34G > C AS primer
AACTGGTGGTGGTTGGAACAC


 


SEQ ID NO: 16
34G > C AS primer
AACTGGTGGTGGTTGGATCAC


 


SEQ ID NO: 17
34G > C AS primer
ATCGGGTGGTGGTTGGAGFAC


 


SEQ ID NO: 18
34G > A AS primer
CAGACTGGTGGTGGTTGGAGFAA


 


SEQ ID NO: 19
34G > A AS primer
AGACTGGTGGTGGTTGGAGCDA


 


SEQ ID NO: 20
34G > A AS primer
AGACTGGTGGTGGTTGGAGFAA


 


SEQ ID NO: 21
35G > T AS primer
AACTGGTGGTGGTTGGAGCAAT


 


SEQ ID NO: 22
35G > T AS primer
AACTGGTGGTGGTTGGAGCATT


 


SEQ ID NO: 23
35G > T AS primer
AACTGGTGGTGGTTGGAGEAAT


 


SEQ ID NO: 24
Exon 2 common
GAATATGGGTAAAGATGATCCGACAA


 


SEQ ID NO: 25
Exon 2 common
GTAAAGATGATCCGACAAGTGAGAGA


 


SEQ ID NO: 26
Exon 2 common
GAATATGGGTAAAGATGATCCGACAAGT


 


SEQ ID NO: 27
Exon 2 probe
JCACTGAECAATCCAGCTAATCCAGAACCACP


 


SEQ ID NO: 28
Exon 2 probe
JCACTGAECAATCCAGCTAATCCAGAACCACP


 


SEQ ID NO: 29
Exon 2 probe
JGTGGTTECTGGATTAGCTGGATTGTCAGTGP





Key:


AS primer: allele-specific primer,


Common: common primer, E = N4-Methyl-dC, M = N6-Methyl-dA, D = N6-tertiary-butyl-benzyl-dA, F = N4-tertiary-butyl-benzyl-dC, J = HEX, Q = BHQ-2, P = Phosphate






The standard PCR mixture included nucleoside triphosphates (including dUTP), DNA polymerase, 0.1 μM each of selective primer, 0.1-0.7 μM common primer, a detection probe, target DNA (9900 copies of wild-type K562 cell line with 100 copies of mutant plasmid, or 10,000 copies of wild-type cell line DNA or 10,000 copies of NRAS wild-type exon 2 or 3 plasmid), and uracil-N-glycosylase. Amplification and analysis were done using the Roche LightCycler® 480 instrument (Roche Applied Science, Indianapolis, Ind.) The following temperature profile was used: 2 cycles of 95° C. (10 seconds) to 62° C. (30 seconds) followed by cycling from 93° C. (10 seconds) to 62° C. (30 seconds) 55 times. Fluorescence data was collected at the start of each 62° C. step in the 55-cycle program.
Results are shown in FIG. 1. Amplification of the wild-type genomic DNA is shown by dashed lines; amplification of the plasmid containing the wild-type sequence is shown by bold solid lines and amplification of the mutant DNA (target sequence) is shown by narrow solid lines. The results demonstrate that when an upstream mutation-specific primer was paired with one of the downstream primers selected from among SEQ ID NOs: 24-26, breakthrough amplification of the non-target (wild-type) sequence was detected. See FIG. 1A (dashed line). When the same mutation-specific primer was paired with a different downstream primer, selected from among SEQ ID NOs: 1-5, breakthrough amplification of the non-target (wild-type) sequence was suppressed, see FIG. 1B. Notably, amplification of the non-target sequence present in a plasmid is unaffected and is not suppressed (bold solid line).
Example 2
Suppression of Breakthrough Amplification by an Additional Suppressor Oligonucleotide
In this example, suppression of breakthrough amplification was observed in an AS-PCR targeting mutations in codon 61 of the human NRAS gene. The primers and probes used in Example 2 are shown in Table 3. An upstream primer selected from among SEQ ID NOs: 30-47 is matched to one of the mutations 183A>T, 183A>C, 181C>A, 182A>T, 182A>C, 182A>G corresponding to amino acid changes Q61Ha, Q61Hb, Q61K, Q61L, Q61P, and Q61R in the human NRAS gene and is mismatched with the wild-type sequence. A downstream primer selected from among SEQ ID NOs: 48-50 and detection probe selected from among SEQ ID NOs: 51-53 are common between the mutant and wild-type sequences in exon 3 of the NRAS gene. Suppressor oligonucleotides selected from among SEQ ID NOs: 1-5 do not hybridize to any of the amplicons defined by the primer pairs used in this example.







TABLE 3







Primers and probes for exon 3 of the NRAS gene used in Example 2.









SEQ ID NO:
Function
Sequence 5′-3′





SEQ ID NO: 30
183A > T AS primer
GGATATACTGGATACAGCTGGACDT


 


SEQ ID NO: 31
183A > T AS primer
GGACATACTGGATACAGCTGGACTT


 


SEQ ID NO: 32
183A > T AS primer
GGACATACTGGATACAGCTGGAGAT


 


SEQ ID NO: 33
183A > C AS primer
ACATACTGGATACAGCTGGACTC


 


SEQ ID NO: 34
183A > C AS primer
ATACTGGATACAGCTGGACTC


 


SEQ ID NO: 35
183A > C AS primer
ATACTGGATACAGCTGGATAC


 


SEQ ID NO: 36
181C > A AS primer
TGGATATACTGGATACAGCTGIAA


 


SEQ ID NO: 37
181C > A AS primer
GACATACTGGATACAGCTGGAA


 


SEQ ID NO: 38
181C > A AS primer
TGGATATACTGGATACAGCTGGMA


 


SEQ ID NO: 39
182A > T AS primer
GAGATACTGGATACAGCTGGAFT


 


SEQ ID NO: 40
182A > T AS primer
GACATACTGGATACAGCTGTACT


 


SEQ ID NO: 41
182A > T AS primer
GACATACTGGATACAGCTGAACT


 


SEQ ID NO: 42
182A > C AS primer
GACGTACTGGATACAGCTGGAFC


 


SEQ ID NO: 43
182A > C AS primer
CGTACTGGATACAGCTGGAFC


 


SEQ ID NO: 44
182A > C AS primer
GACATACTGGATACAGCTGAACC


 


SEQ ID NO: 45
182A > G AS primer
GACATACTGGATACAGCTGGTEG


 


SEQ ID NO: 46
182A > G AS primer
ACGTACTGGATACAGCTGGAFG


 


SEQ ID NO: 47
182A > G AS primer
GACACACTGGATACAGCTGGAFG


 


SEQ ID NO: 48
Exon 3 common
AGAGAAAATAATGCTCCTAGTACCTGTAG


 


SEQ ID NO: 49
Exon 3 common
TCCTTTCAGAGAAAATAATGCTCCTAGT


 


SEQ ID NO: 50
Exon 3 common
GTTAATATCCGCAAATGACTTGCTATTATT


 


SEQ ID NO: 51
Exon 3 probe
JCTGTCCETCATGTATTGGTCTCTCATGGCACTGP


 


SEQ ID NO: 52
Exon 3 probe
JCTCATGETATTGGTCTCTCATGGCACTGTACP


 


SEQ ID NO: 53
Exon 3 probe
JCTTCGCECTGTCCTCATGTATTGGTCTCTCP





Key:


AS primer: allele-specific primer,


Common: common primer, E = N4-Methyl-dC, M = N6-Methyl-dA, D = N6-tertiary-butyl-benzyl-dA, F = N4-tertiary-butyl-benzyl-dC, I = Inosine, J = FAM, Q = BHQ-2, P = Phosphate






In this example, the same reaction conditions were used as in Example 1, except in addition to the upstream and downstream primer, one of the suppressor oligonucleotides selected from among SEQ ID NOs: 1-5 was added to the reaction at 0.1 or 0.7 μM.
Results are shown in FIG. 2. Amplification of the wild-type genomic DNA is shown by dashed lines and amplification of the mutant DNA (target sequence) is shown by narrow solid lines. The results demonstrate that when the primer pair composed of a common primer and a Q61 mutation-specific primer was used, breakthrough amplification of the non-target sequences was detected. See FIG. 2A (dashed lines). When the suppressor oligonucleotide was also present in the reaction mixture at 0.1 μM, breakthrough amplification of the non-target sequences was not suppressed, see FIG. 2B. But when the suppressor oligonucleotide was present in the reaction mixture at 0.7 μM, breakthrough amplification of the non-target sequences was suppressed, see FIG. 2C. In this example, all the primers are present at 0.1 μM while the suppressing oligonucleotide was present either at 0.1 μM or 0.7 μM.
Example 3
Suppression of Breakthrough Amplification of the Unrelated Template PI3KCA by a Suppressor Oligonucleotide
In this example, suppression of breakthrough amplification was observed in an AS-PCR targeting mutations in codon 1049 of the human PI3KCA gene. The primers and probes used in Example 3 are shown in Table 4. An upstream primer selected from among SEQ ID NOs: 54-56 is matched to the mutation 3145G>C corresponding to the amino acid change G1049R in the human PI3KCA gene and is mismatched with the wild-type sequence. A downstream primer selected from among SEQ ID NOs: 57-59 and a probe selected from among SEQ ID NOs: 60 & 96 and 61 & 97 are common between the mutant and wild-type sequences. Suppressor oligonucleotides selected from among SEQ ID NOs: 1-5 (specific for the human NRAS gene) do not hybridize to the PI3KCA amplicons used in this example.







TABLE 4







Primers and probes for the PI3KCA gene used in Example 3.









SEQ ID NO:
Function
Sequence 5′-3′





SEQ ID NO: 54
3145G > C AS primer
CATGAAACAAATGAATGATGCACATCCTC


 


SEQ ID NO: 55
3145G > C AS primer
CATGAAACAAATGAATGATGCACATCGTC


 


SEQ ID NO: 56
3145G > C AS primer
CATGAAACAAATGAATGATGCACATTATC


 


SEQ ID NO: 57
3145 common
CAATGCATGCTGTTTAATTGTGTGGA


 


SEQ ID NO: 58
3145 common
TTCAGTTCAATGCATGCTGTTTAATTGTG


 


SEQ ID NO: 59
3145 common
GTGGAATCCAGAGTGAGCTTTCAT


 


SEQ ID NOS
3145 probe
JTGGCTGGACAAQCAAAAATGGATTGGATCP


60 and 96




 


SEQ ID NOS
3145 probe
JATGGATTGGAQTCTTCCACACAATTAAACAGCATGP


61 and 97





KEY


AS primer: allele-specific primer,


Common: common primer, J = JA270, Q = BHQ-2, P = Phosphate






In this example, the same reaction conditions were used as in Example 1, except in addition to the upstream and downstream primer, one of the suppressor oligonucleotides selected from among SEQ ID NOs: 1-5 was added to the reaction at 1.0 μM.
Results are shown in FIG. 3. Amplification of the wild-type genomic DNA is shown by dashed lines; and amplification of the mutant DNA (target sequence) is shown by narrow solid lines. The results demonstrate that when the primer pair composed of a G1049R-specific primer and a common primer was used, breakthrough amplification of the non-target (wild-type) sequence was detected. See FIG. 3A (dashed lines). When the suppressor oligonucleotide selected from among SEQ ID NOs: 1-5 was also present in the reaction mixture, breakthrough amplification of the non-target (wild-type) sequence was suppressed, with no impact on the specific amplification of the target (mutant G1049R) sequence (solid lines). See FIG. 3B. The same suppressing oligonucleotide selected from among SEQ ID NOs: 1-5 was also added to allele-specific PCR designed to detect PI3KCA mutations 1258T>C, 1635G>T, 1634A>G, and 1633G>A. The same trend was observed: no impact on specific amplification of the target (mutant) sequence and suppression of the breakthrough amplification of the non-target (wild-type) sequence (data not shown).
Example 4
Suppression of Breakthrough Amplification of the Unrelated Template BRAF by a Suppressor Oligonucleotide
In this example, partial suppression of breakthrough amplification was observed in an AS-PCR targeting mutations in codons 469 and 600 of the human BRAF gene. The primers and probes used in Example 4 are shown in Table 5. For mutations in codon 469, the upstream primer was selected from among SEQ ID NOs: 62-70. These primers are matched to various mutations at codon 469 in exon 11. For mutations in codon 600, the upstream primer was selected from among SEQ ID NOs: 75-86. These primers are matched to various mutations at codon 600 in exon 15. For the codon 469 mutations, the common downstream primer was selected from among SEQ ID NOs: 71-72, and the probe was selected from among SEQ ID NOs: 73 & 98 and 74 & 99. For the codon 600 mutations, the downstream primer was selected from among SEQ ID NOs: 87-89, and the probe was selected from among SEQ ID NOs: 90-92. Suppressor oligonucleotides selected from among SEQ ID NOs: 1-5 (specific for the human NRAS gene) do not hybridize to the BRAF amplicons defined by any of the primer pairs used in this example.







TABLE 5







Primers and probes for the BRAF gene used in the Example 4.









SEQ ID NO:
Function
Sequence 5′-3′





SEQ ID NO: 62
1406G > C AS primer
AAAGAATTGGATCTGGATCATTAGC


 


SEQ ID NO: 63
1406G > C AS primer
AAAGAATTGGATCTGGATCATTCGC


 


SEQ ID NO: 64
1406G > C AS primer
AAAGAATTGGATCTGGATCATGTGC


 


SEQ ID NO: 65
1405G > A AS primer
AAAGAATTGGATCTGGATCATATA


 


SEQ ID NO: 66
1405G > A AS primer
ACAAAGAATTGGATCTGGATCATTAA


 


SEQ ID NO: 67
1406G > T AS primer
AGTGGGACAAAGAATTGGATCAGT


 


SEQ ID NO: 68
1406G > T AS primer
AGTGGGACAAAGAATTGGATCTAT


 


SEQ ID NO: 69
1406G > A AS primer
ACAAAGAATTGGATCTGGATCATTTAA


 


SEQ ID NO: 70
1406G > A AS primer
GACAAAGAATTGGATCTGGATCATTTAA


 


SEQ ID NO: 71
Exon 11 Common
GCGAACAGTGAATATTTCCTTTGATG


 


SEQ ID NO: 72
Exon 11 Common
GACTTGTCACAATGTCACCACATTACATA


 


SEQ ID NOS
Exon 11 Probe
EAGTCTACAAGQGGAAAGTGGCATGGTAAP


73 and 98




 


SEQ ID NOS
Exon 11 Probe
ETGGCATGGTAQAGTATGTAATGTGGTGACATTP


74 and 99




 


SEQ ID NO: 75
1798_1799GT > AA,
AGTAAGAATAGGTGATTTTGGTCTAGCTACFA



1798_1799GT > AG,




1798G > A AS primer



 


SEQ ID NO: 76
1798_1799GT > AA,
AGTAAGAATAGGTGATTTTGGTCTAGCTALAA



1798_1799GT > AG,




1798G > A AS primer



 


SEQ ID NO: 77
1798_1799GT > AA,
AGTAAGAATAGGTGATTTTGGTCTAGCTCLAA



1798_1799GT > AG,




1798G > A AS primer



 


SEQ ID NO: 78
1798G > T AS primer
AGTAAGAATAGGTGATTTTGITCTAGCTACFT


 


SEQ ID NO: 79
1798G > T AS primer
AGTAAGAATAGGTGATTTTGGTCTAICTACFT


 


SEQ ID NO: 80
1798G > T AS primer
AGTAAGAATAGGTGATTTTGGTCTAGCTACFT


 


SEQ ID NO: 81
1799T > G AS primer
AATGGGTGATTTTGGTCTAGCTFCTGG


 


SEQ ID NO: 82
1799T > G AS primer
AATGGGTGATTTTGGTCTAGCTFTAIG


 


SEQ ID NO: 83
1799T > G AS primer
AGTAGGTGATTTTGGTCTAGCTATFGG


 


SEQ ID NO: 84
1799T > C AS primer
AATGGGTGATTTTGGTCTAGCTFTAIC


 


SEQ ID NO: 85
1799T > C AS primer
AATGGGTGATTTTGGTCTAGCTALTIC


 


SEQ ID NO: 86
1799T > C AS primer
AATGGGTGATTTTGGTCTAGCTALTGC


 


SEQ ID NO: 87
Exon 15 Common
GTGGAAAAATAGCCTCAATTCTTACCA


 


SEQ ID NO: 88
Exon 15 Common
TAGCCTCAATTCTTACCATCCACAAAA


 


SEQ ID NO: 89
Exon 15 Common
CTAGTAACTCAGCAGCATCTCAG


 


SEQ ID NO: 90
Exon 15 Probe
ETGGATCQCAGACAACTGTTCAAACTGATGGGP


 


SEQ ID NO: 91
Exon 15 Probe
ETCCCATQCAGTTTGAACAGTTGTCTGGATCCAP


 


SEQ ID NO: 92
Exon 15 Probe
ETCTCGATGGAGTGGGTCCQP





KEY


AS primer: allele-specific primer,


Common: common primer, F = N6-tertiary-butyl-benzyl-dA, L = N4-tertiary-butyl-benzyl-dC, I = Inosine, E = FAM, Q = BHQ-2, P = Phosphate






In this example, the same reaction conditions were used as in Example 3.
Results are shown in FIG. 4. Amplification of the wild-type genomic DNA is shown by dashed lines and amplification of the BRAF codon 469 and 600 targets is shown by solid lines. The results demonstrate that when the primer pair consisting of a primer matched to one of the codon 469 mutations and a common primer was used, breakthrough amplification of the non-target (wild-type) sequence was detected, see FIG. 4A (dashed lines). When a suppressor oligonucleotide selected from among SEQ ID NOS: 1-5 was also present in the reaction mixture, breakthrough amplification of the non-target (wild-type) sequence was suppressed (dashed lines,) with slight impact on the specific amplification of the mutant sequence (solid lines). See FIG. 4B. When the primer pair consisting of a primer matched to one of the codon 600 mutations and a common primer was used, breakthrough amplification of the non-target (wild-type) sequence was detected. See FIG. 4C (dashed lines). When a suppressor oligonucleotide selected from among SEQ ID NOs: 1-5 was also present in the reaction mixture, breakthrough amplification of the non-target (wild-type) sequence was partially suppressed see FIG. 4D. Incomplete suppression of the non-target amplification and slight impact on the target amplification observed with the BRAF system suggests that the suppression phenomenon may be sequence-dependent.
Example 5
Suppression of Breakthrough Amplification by Linear Primer Extension Reactions
In this example, suppression of breakthrough amplification of the NRAS template was observed in the presence of the M13 template and a series of M13-specific primers. The AS-PCR targeted mutations in codons 12 and 61 of the human NRAS gene. The M13 primers used in Example 5 are shown in Table 6. For the NRAS target, the upstream primer was selected from among SEQ ID NOs: 30-47. These primers are matched to one of the mutations 183A>T, 183A>C, 181C>A, 182A>T, 182A>C, 182A>G corresponding to amino acid changes Q61Ha, Q61Hb, Q61K, Q61L, Q61P, and Q61R in the human NRAS gene and are mismatched with the wild-type sequence. The downstream primer selected from among SEQ ID NOs: 48-50 and the probe selected from among SEQ ID NOs: 51-53 are common between the mutant and wild-type sequences of exon 3 in the human NRAS gene. The upstream primer selected from among SEQ ID NOs: 6-23, is matched to one of the mutations 35G>C, 34G>T, 35G>A, 34G>C, 34G>A, and 35G>T in exon 2 of the human NRAS gene and is mismatched with the wild-type sequence. The downstream primer selected from SEQ ID NOs: 24-26 and the detection probe selected from SEQ ID NOs: 27-29 are common between the mutant and wild-type sequences of exon 2 in the human NRAS gene. The reaction mixture also contained single-stranded circular DNA of bacteriophage M13 and three primers (SEQ ID NOs: 93-95, Table 6) oriented in the same direction to ensure linear amplification of the viral template.







TABLE 6







M13 primers used Example 5.









SEQ ID NO:
Function
Sequence 5′-3′





SEQ ID NO: 93
M13 Primer
ACATGAAAGTATTAAGAGGCTGAGA




CTCCTCA


 


SEQ ID NO: 94
M13 Primer
GAAGAAAGCGAAAGGAGCGGGC


 


SEQ ID NO: 95
M13 Primer
GGAACGAGGGTAGCAACGGCTACA









In this example, the same reaction conditions were used as in Example 1, except the M13 single stranded bacteriophage template was added at 10,000 copies per reaction, and primers, SEQ ID NOs: 63-65, were added at equimolar concentrations of 0.033 μM each for a total concentration of 0.1 μM.
Results are shown in FIG. 5. Amplification of the wild-type genomic DNA is shown by dashed lines and amplification of the NRAS codon 12 or codon 61 mutant targets is shown by solid lines. The results demonstrate that when the primer pair consisting of an allele-specific primer matched to one of the mutations in codon 61 and a common primer was used, breakthrough amplification of the non-target (wild-type) NRAS sequence was detected. See FIG. 5A (dashed lines). When the M13 DNA and the three primers capable of linear amplification of the M13 DNA were also present in the reaction mixture, breakthrough amplification of the non-target (wild-type) NRAS sequence was suppressed. See FIG. 5B. By comparison, when the primer pair consisting of an allele-specific primer matched to one of the mutations in codon 12 and a common primer was used, breakthrough amplification of the non-target (wild-type) NRAS sequence was detected. See FIG. 5C (dashed lines). This breakthrough amplification was not suppressed by the M13 DNA and the three primers capable of linear amplification. See FIG. 5D.
Example 6
Breakthrough Suppression by Suppressor Oligonucleotides with Varying Degrees of Homology to the Target Genome
In this example, suppression of breakthrough amplification with several suppressor oligonucleotides was observed in an AS-PCR targeting mutations in codon 12 of the human NRAS gene. An upstream primer was selected from among SEQ ID NOs: 6-23, the primers matched to one of the codon 12 mutations (35G>C, 34G>T, 35G>A, 34G>C, 34G>A, and 35G>T, corresponding to amino acid changes G12A, G12C, G12D, G12R, G12S, and G12V) in the human NRAS gene and mismatched with the wild-type sequence. The upstream primer was paired with different downstream primers acting as suppressors of breakthrough amplification. These downstream primers represented by SEQ ID NOs: 1-5 and 24-26, have varying degrees of homology to the target genome ranging between low, medium and high as determined according to the method of the present invention. See Example 8 and FIG. 8.
In this example, the same reaction conditions were used as in Example 1. The suppressor oligonucleotides with low, medium and high homology were used at 0.1 μM.
Results are shown in FIG. 6. Amplification of the wild-type genomic DNA is shown by dashed lines; amplification of the NRAS codon 12 targets is shown by solid lines. The results demonstrate that the downstream primer with the highest degree of homology to the target genome as determined by the method of the present invention (SEQ ID NO: 1), produced the highest level of suppression (See FIG. 6C), while the downstream primers with the medium degree of homology (SEQ ID NOs: 2-5) produced a lower level of suppression, see FIG. 6B. The downstream primers with the lowest degree of homology (SEQ ID NOs: 24-26) had no effect on wild-type breakthrough and showed no suppression, see FIG. 6A. It is also worth noting that the suppressing oligonucleotides (SEQ ID NOs: 1-5) had varying degrees of suppression, but had no negative impact on the specific amplification as measured by Ct.
Example 7
Selecting Regions of Homology within the Region of Interest
In this example, human NRAS gene was selected as the region of interest for designing suppressor oligonucleotides. The 488 base pair region from exon 2 of the NRAS gene was used as a query sequence to be compared to the human genome sequence under relaxed conditions selecting the option that finds “somewhat similar sequences” using the algorithm “blastn.” FIG. 7 shows that the search revealed regions of multiple homologies in the portions defined by nucleotides 180-270 and 360-450 of the query sequence. These regions were selected as regions of interest for design of suppressor oligonucleotides.
Example 8
Selecting Suppressor Oligonucleotides from the Region of Interest
In this example, several oligonucleotides from the regions of interest were designed and subjected to a BLAST® analysis to determine regions of homology in the human genome meeting the criteria set forth by the present invention. FIG. 8 shows parameters for each oligonucleotide and the actual ability to suppress breakthrough amplification in reactions. The parameters include the length of the oligonucleotide under the column “nMer”. Under the column “Total Hits” is the total number of “Blast Hits” between the oligonucleotide and the target genome with the program Blastn was able to find. The program stringency was set on “somewhat similar sequences”. Under the column “Hits with Criteria”, this is the total number of hits that meet the criteria of 75% identity and fewer than two mismatches at the 3′ terminus. The column “Degree of Homology” contains a value assigned as follows: the degree of homology to the target genome was said to be “low” when there was only one hit that meets the criteria set forth by the present invention, the degree of homology was said to be “medium” when there were ten or fewer hits that meet the criteria, and the degree of homology was said to be “high” when there were more than 10 hits that meet the criteria. Lastly, the “Breakthrough” column indicates whether or not breakthrough amplification was observed in the presence of the oligonucleotide.
While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

## Claims

1. A reaction mixture for performing an amplification reaction of a target sequence in the genome of a target organism with reduced amplification of non-target sequences, comprising:
at least one allele-specific primer specific for a variant of the target sequence;
an amplicon formed by the at least on allele-specific primer and a reverse primer;
a suppressor oligonucleotide (a) having no significant similarity with the target sequence as determined by the blastn algorithm for somewhat similar sequences, (b) extendable by a DNA polymerase, and (c) having a sequence comprising at least one region of complementarity to multiple sites in the genome of the target organism, wherein the least one region of complementarity: (i) is at least 15 base pairs long; and (ii) spans the 3′-end of the suppressor oligonucleotide; and
a fluorescently labeled probe oligonucleotide, wherein the reaction mixture does not include an amplicon formed by the suppressor oligonucleotide.

2. The reaction mixture of claim 1, wherein the suppressor oligonucleotide is selected from a group consisting of SEQ ID NOs: 1-3, and 5.

3. A reaction mixture for performing an amplification reaction of a target sequence in the genome of a target organism with reduced amplification of non-target sequences, comprising:
at least one allele-specific primer specific for a variant of the target sequence comprising at least one unconventional-base nucleotide;
an amplicon formed by the at least one allele-specific primer and a reverse primer;
a suppressor oligonucleotide (a) having no significant similarity with the target sequence as determined by the blastn algorithm for somewhat similar sequences, (b) extendable by a DNA polymerase, and (c) having a sequence comprising at least one region of complementarity to multiple sites in the genome of the target organism, wherein the least one region of complementarity: (i) is at least 15 base pairs long; and (ii) spans the 3′-end of the suppressor oligonucleotide;
buffer necessary for the function of a DNA polymerase; wherein the reaction mixture does not include an amplicon formed by the suppressor oligonucleotide.

4. The reaction mixture of claim 3, wherein the suppressor oligonucleotide is selected from a group consisting of SEQ ID NOs: 1-3, and 5.

5. The reaction mixture of claim 3, wherein said unconventional-base nucleotide is alkylated.

6. The reaction mixture of claim 1, further comprising nucleotide triphosphates, a DNA polymerase, and a buffer necessary for the function of the polymerase.

7. The reaction mixture of claim 1, further comprising nucleotide triphosphates, a DNA polymerase, and a buffer necessary for the function of the polymerase.