Methods for detecting nucleic acid sequence variations

This application is a continuation application of U.S. Ser. No. 10/202,896, filed Jul. 26, 2002, which is a continuation-in-part of U.S. Ser. No. 09/894,788 filed Jun. 28, 2001, which is a divisional of U.S. Ser. No. 09/590,061 filed Jun. 8, 2000, now U.S. Pat. No. 6,316,200, issued Nov. 13, 2001, and is a continuation-in-part of U.S. Ser. No. 09/335,218, the entire contents of which are incorporated by reference herein.

The present invention relates to oligonucleotides and methods for amplifying and detecting sequence variations in target nucleic acids such as the human β₂-adrenergic receptor (β₂AR) gene. The preferred method involves using fluorescent real-time thermophilic Strand Displacement Amplification (SDA) with nucleic acid primers and adapter-mediated universal detector probes to amplify and detect allele-specific sequences from blood, tissue and bodily fluids.

Sequence-specific hybridization of labeled oligonucleotide probes has long been used as a means for detecting and identifying selected nucleotide sequences, and labeling of such probes with fluorescent labels has provided a relatively sensitive, nonradioactive means for facilitating detection of probe hybridization. Recently developed detection methods employ the process of fluorescence energy transfer (FET) rather than direct detection of fluorescence intensity for detection of probe hybridization. Fluorescence energy transfer occurs between a donor fluorophore and a quencher dye (which may or may not be a fluorophore) when the absorption spectrum of one (the quencher) overlaps the emission spectrum of the other (the donor) and the two dyes are in close proximity. Dyes with these properties are referred to as donor/quencher dye pairs or energy transfer dye pairs. The excited-state energy of the donor fluorophore is transferred by a resonance dipole-induced dipole interaction to the neighboring quencher. This results in quenching of donor fluorescence. In some cases, if the quencher (also referred to as an “acceptor”) is also a fluorophore, the intensity of its fluorescence may be enhanced. The efficiency of energy transfer is highly dependent on the distance between the donor and quencher, and equations predicting these relationships have been developed by Förster (1948. Ann. Phys. 2, 55-75). The distance between donor and quencher dyes at which energy transfer efficiency is 50% is referred to as the Förster distance (R_O). Other mechanisms of fluorescence quenching are also known including, for example, charge transfer and collisional quenching. In these cases the quencher may be a fluorescent dye but it need not be. Fluorescence quenching mechanisms that are not based on FET typically do not require appreciable overlap between the absorption spectrum of the quencher and the emission spectrum of the donor fluorophore.

Energy transfer and other mechanisms which rely on the interaction of two dyes in close proximity to produce quenching are an attractive means for detecting or identifying nucleotide sequences, as such assays may be conducted in homogeneous formats. Homogeneous assay formats are simpler than conventional probe hybridization assays which rely on detection of the fluorescence of a single fluorophore label, as heterogeneous assays generally require additional steps to separate hybridized label from free label. Typically, FET and related methods have relied upon monitoring a change in the fluorescence properties of one or both dye labels when they are brought together by the hybridization of two complementary oligonucleotides. In this format, the change in fluorescence properties may be measured as a change in the amount of energy transfer or as a change in the amount of fluorescence quenching, typically indicated as an increase in the fluorescence intensity of one of the dyes. In this way, the nucleotide sequence of interest may be detected without separation of unhybridized and hybridized oligonucleotides. The hybridization may occur between two separate complementary oligonucleotides, one of which is labeled with the donor fluorophore and one of which is labeled with the quencher. In double-stranded form there is decreased donor fluorescence (increased quenching) and/or increased energy transfer as compared to the single-stranded oligonucleotides. Several formats for FET hybridization assays are reviewed in Nonisotopic DNA Probe Techniques (1992. Academic Press, Inc., pgs. 311-352). Alternatively, the donor and quencher may be linked to a single oligonucleotide such that there is a detectable difference in the fluorescence properties of one or both when the oligonucleotide is unhybridized vs. when it is hybridized to its complementary sequence. In this format, donor fluorescence is typically increased and energy transfer/quenching are decreased when the oligonucleotide is hybridized. For example, an oligonucleotide labeled with donor and quencher dyes may contain self-complementary sequences that base-pair to form a hairpin which brings the two dyes into close spatial proximity where energy transfer and quenching can occur. Hybridization of this oligonucleotide to its complementary sequence in a second oligonucleotide disrupts the hairpin and increases the distance between the two dyes, thus reducing quenching. See Tyagi and Kramer (1996. Nature Biotech. 14, 303-308) and B. Bagwell, et al. (1994. Nucl. Acids Res. 22, 2424-2425; U.S. Pat. No. 5,607,834). Homogeneous methods employing energy transfer or other mechanisms of fluorescence quenching for detection of nucleic acid amplification have also been described. L. G. Lee, et al. (1993. Nuc. Acids Res. 21, 3761-3766) disclose a real-time detection method in which a doubly-labeled detector probe is cleaved in a target amplification-specific manner during PCR. The detector probe is hybridized downstream of the amplification primer so that the 5′-3′ exonuclease activity of Taq polymerase digests the detector probe, separating two fluorescent dyes which form an energy transfer pair. Fluorescence intensity increases as the probe is cleaved. Signal primers (sometimes also referred to as detector probes) which hybridize to the target sequence downstream of the hybridization site of the amplification primers have been described for homogeneous detection of nucleic acid amplification (U.S. Pat. No. 5,547,861 which is incorporated herein by reference). The signal primer is extended by the polymerase in a manner similar to extension of the amplification primers. Extension of the amplification primer displaces the extension product of the signal primer in a target amplification-dependent manner, producing a double-stranded secondary amplification product which may be detected as an indication of target amplification. Examples of homogeneous detection methods for use with single-stranded signal primers are described in U.S. Pat. No. 5,550,025 (incorporation of lipophilic dyes and restriction sites) and U.S. Pat. No. 5,593,867 (fluorescence polarization detection). More recently signal primers have been adapted for detection of nucleic acid targets using FET/fluorescence quenching methods which employ unfolding of secondary structures (e.g., U.S. Pat. No. 5,691,145 and U.S. Pat. No. 5,928,869). Partially single-stranded, partially double-stranded signal primers labeled with donor/quencher dye pairs have also recently been described. For example, U.S. Pat. No. 5,846,726 discloses signal primers with donor/quencher dye pairs flanking a single-stranded restriction endonuclease recognition site. In the presence of the target, the restriction site becomes double-stranded and cleavable by the restriction endonuclease. Cleavage separates the dye pair and decreases donor quenching. U.S. Pat. No. 6,130,047 (incorporated herein by reference) describes a detector nucleic acid comprised of two complementary oligonucleotides that are hybridized to form a duplex. One of the oligonucleotides is longer than the other and contains a single-stranded tail sequence capable of binding target sequences. The two oligonucleotides also comprise a fluorophore/quencher dye pair such that when the two oligonucleotides are hybridized to each other fluorescence remains substantially quenched, because fluorophore and quencher remain in close spatial proximity. Hybridization of a target sequence to the single-stranded tail of the longer oligonucleotide enables a polymerase-mediated displacement of the shorter oligonucleotide from the longer one, resulting in separation of quencher from fluorophore and a corresponding increase in fluorescence of the sample.

U.S. Pat. No. 6,379,888 (incorporated herein by reference) also discloses a signal primer comprised of two complementary oligonucleotides that are hybridized to form a duplex with one of the oligonucleotides containing in addition a single-stranded tail capable of binding target sequences. In this case, however, the shorter of the two oligonucleotides contains both a fluorophore and a quencher which are held spatially apart when the shorter oligonucleotide is hybridized to the longer, unlabeled oligonucleotide. Hybridization of a target sequence to the single-stranded tail of the longer oligonucleotide triggers a polymerase-mediated displacement of the shorter oligonucleotide. Upon displacement, the shorter oligonucleotide adopts a conformation that brings the fluorophore and quencher into close proximity so fluorescence decreases in the presence of target. U.S. Pat. No. 5,866,336 describes use of a fluorescently labeled hairpin on an amplification primer in PCR. The 3′ end of the hairpin primer hybridizes to the complement of a non-target sequence appended to the target by a second primer. In this system, the hairpin primer plays an integral part in amplification of the target sequence and must be extendible. In contrast, in the present invention it is not necessary for the reporter probe to be extendible, as it does not participate in amplification of the target sequence but generates signal in a separate series of reaction steps which occur concurrently with target amplification. In further contrast, the signal primers of the invention hybridize to an internal sequence of the target (i.e., between the amplification primers), so that the signal generation reaction detects a subsequence of the target, not the amplification product itself.

Detecting and identifying variations in DNA sequences among individuals and species has provided insights into evolutionary relationships, inherited disorders, acquired disorders and other aspects of molecular genetics including predisposition to infectious or non-infectious disease and prediction of therapeutic efficacy. Analysis of sequence variation has routinely been performed by analysis of restriction fragment length polymorphism (RFLP) which relies on a change in restriction fragment length as a result of a change in sequence. RFLP analysis requires size-separation of restriction fragments on a gel and Southern blotting with an appropriate probe. This technique is slow and labor intensive and cannot be used if the sequence change does not result in a new or eliminated restriction site.

More recently, PCR has been used to facilitate sequence analysis of DNA. For example, allele-specific oligonucleotides have been used to probe dot blots of PCR products for disease diagnosis. If a point mutation creates or eliminates a restriction site, cleavage of PCR products may be used for genetic diagnosis (e.g., sickle cell anemia). General PCR techniques for analysis of sequence variations have also been reported. S. Kwok, et al. (1990. Nucl. Acids Res. 18:999-1005) evaluated the effect on PCR of various primer-template mismatches for the purpose of designing primers for amplification of HIV which would be tolerant of sequence variations. The authors also recognized that their studies could facilitate development of primers for allele-specific amplification. Kwok, et al. report that a 3′ terminal mismatch on the PCR primer produced variable results. In contrast, with the exception of a 3′ T mismatch, a 3′ terminal mismatch accompanied by a second mismatch within the last four nucleotides of the primer generally produced a dramatic reduction in amplification product. The authors report that a single mismatch one nucleotide from the 3′ terminus (N−1), two nucleotides from the 3′ terminus (N−2) or three nucleotides from the 3′ terminus (N−3) had no effect on the efficiency of amplification by PCR. C. R. Newton, et al. (1989. Nucl. Acids Res. 17:2503-2516) report an improvement in PCR for analysis of any known mutation in genomic DNA. The system is referred to as Amplification Refractory Mutation System or ARMS and employs an allele-specific PCR primer. The 3′ terminal nucleotide of the PCR amplification primer is allele specific and therefore will not function as an amplification primer in PCR if it is mismatched to the target. The authors also report that in some cases additional mismatches near the 3′ terminus of the amplification primer improve allele discrimination.

The present invention provides methods for identifying sequence variations in a nucleic acid sequence of interest using an unlabeled signal primer comprising a 5′ adapter sequence to mediate detection by genetic or universal labeled reporter probes. The method is based upon the universal detection system described in U.S. Pat. No. 6,316,200 (herein incorporated by reference) (FIG. 1A, B). The 3′ end of the reporter probe hybridizes to the complement of the 5′ adapter sequence to produce a 5′ overhang. Polymerase is used to fill in the overhang and synthesize the compliment of the 5′ overhang of the reporter probe. Synthesis of the reporter probe compliment is detected, directly or indirectly, as an indication of the presence of the specific target allele.

The 5′ tail sequence of the signal primer comprises a sequence which does not hybridize to the target (the adapter sequence). The adapter sequence may be selected such that it is the same in a variety of signal primers which have different 3′ target binding sequences (i.e., a “universal” 5′ tail sequence). This allows a single reporter probe sequence to be used for detection of any desired target sequence, which is an advantage in that synthesis of the reporter probe is more complex due to the labeling. Further, the invention simplifies the synthesis of the target-specific signal primer. As the signal primer is not labeled, signal primers with different target binding sequences specific for different targets may be more easily and efficiently synthesized. The methods of the invention therefore permit the detection of many different mutations using a single pair of detectable reporter probes and this offers a particular advantage over other systems that use target-specific reporter probes for the detection of allelic variations. The present invention offers significant benefits over such techniques in terms of cost and speed of development of novel assays.

The methods of the invention are particularly well suited, but are not limited to, the detection and identification of single nucleotide differences between the target sequence being evaluated (e.g., a mutant allele of a gene) and a second nucleic acid sequence (e.g., a wild-type allele for the same gene), as they make use of nucleotide mismatches near the 3′ end of the signal primer to discriminate between a first nucleotide and a second nucleotide at the site of interest in the target. Both the wild-type and mutant alleles can be detected in the same reaction by incorporating signal primers specific for each target (FIG. 2A, B). In a preferred embodiment, the diagnostic nucleotide (SNP-site) is located one base (N−1) from the 3′ terminus of the signal primer. This reduces the efficiency of non-specific polymerase extension by reducing the stability of base pairing and base stacking interactions at the 3′ end of the signal primer. A further embodiment of the invention involves the creation of artificial mismatches in the signal primer sequence at one or more nucleotides (e.g., N−2, N−3, N−4, and N−5) near the SNP-site N−1). This further reduces the stability of hybridization at the 3′ end of the signal primer and lowers the melting temperature of the primer:target hybrid. This embodiment has no impact on the amplification efficiency of the target nucleic acid as this occurs independently of hybridization of the signal primer. However, the efficiency of detection, particularly that of a target sequence containing multiple mismatches with the signal primer is diminished, thereby enhancing allelic discrimination. This may be of particular importance in systems designed to discriminate sequence variations located in G-C rich regions of DNA and in which base pairing and base stacking interactions are very strong. Such mismatches may also be introduced in the signal primer downstream of the diagnostic nucleotide (e.g., at positions δ+1, δ+2, δ+3 or δ8+4 relative to the diagnostic nucleotide, δ) to bring about a similar reduction in the efficiency of polymerase extension. The disclosed methods have distinct advantages over other primer extension-based systems for allelic discrimination in which the diagnostic nucleotide is incorporated in an amplification primer. In the method of the present invention, multiple mutations can be detected within the target sequence using the same amplification primers in conjunction with unlabeled signal primers that are specific for each mutation. This obviates the need to design and optimize multiple amplification systems for the detection of each individual mutation.

In a preferred embodiment, the method of the invention employs Strand Displacement Amplification (SDA) as the means of target amplification. SDA relies upon the coordinated activity of a DNA polymerase and restriction enzyme to amplify target nucleic acid. A limitation of SDA therefore, is that the target sequence ideally should not contain the SDA restriction enzyme recognition site. For many applications, this limitation can be overcome through careful selection of the target region. However, for SNP analysis in which a specific mutation at a particular site must be identified, it is not always possible to avoid undesirable restriction sites. To overcome this obstacle, artificially created mismatches in bumper and amplification primer sequences can be used to protect the amplicon from the digestion by the restriction enzyme used in SDA (FIGS. 3A and B).

In the isothermal amplification methods of the present invention a mismatch on the detector/amplification primer at N−1 to N−4 and a complementary 3′ terminal nucleotide results in excellent allele discrimination, particularly if an optional second nondiagnostic mismatch is included. This embodiment is therefore preferred for detector/amplification primers of the invention.

In an alternative preferred embodiment, the detector primer is used in an isothermal amplification reaction as a signal primer (also referred to as a detector probe) as taught in U.S. Pat. No. 5,547,861, the disclosure of which is hereby incorporated by reference. In the amplification reaction, the signal primer hybridizes to the target sequence downstream of an amplification primer such that extension of the amplification primer displaces the signal primer and its extension product. After extension, the signal primer includes the downstream sequence which is the hybridization site for the second amplification primer. The second amplification primer hybridizes to the extended signal primer and primes synthesis of its complementary strand. Production of these double-stranded secondary amplification products may be detected not only as an indication of the presence of the target sequence, but in the methods of the invention a signal primer which has the sequence characteristics of a detector primer (a detector/signal primer) also facilitates detection and/or identification of SNP\'s within the target sequence. In this embodiment, a diagnostic mismatch at either the 3′ terminus (N) or at N−1 to N−4 provides excellent allele discrimination.

Applicants hypothesize that the different results obtained with a diagnostic mismatch at the 3′ terminus of a detector/signal primer as compared to a diagnostic mismatch at the 3′ terminus of a detector/amplification primer may be at least partially due to a kinetic effect. If a signal primer is not efficiently extended on a target to which it is hybridized (e.g., when it contains mismatches), it will be quickly displaced from the template by extension of the upstream amplification primer. If the signal primer is efficiently extended, extension will occur before the signal primer is displaced from the target. That is, the upstream amplification primer (which is typically perfectly matched and efficiently extended) imposes a “time-limit” for extension on the detector/signal primer. In contrast, the amplification primer in an isothermal amplification reaction does not have a time-limit for extension imposed upon it by additional components of the isothermal amplification reaction or by thermocycling. Therefore, with sufficient time available, a detector/amplification primer may eventually be extended even when the extension reaction is inefficient. This phenomenon could reduce discrimination between alleles when a detector/amplification primer with a 3′ terminal mismatch is employed in isothermal amplification reactions. In addition, the ability of amplification primers to correct a mismatch with the target may contribute to these observations. Amplification primers produce amplicons that are perfectly matched with the amplification primers which produced them, thus eliminating the basis of allele discrimination. In contrast, such “correction” does not occur with signal primers.

Another embodiment uses signal primers with target binding sequences that are at least partially identical to the target binding sequence of an amplification primer (FIG. 4). Competitive hybridization between two oligonucleotides in an amplification/detection system has been described previously (U.S. Pat. No. 6,258,546 herein incorporated by reference) for qualitative and quantitative detection of nucleic acids. This approach provides detection efficiency that is equal to or better than that of conventional signal primers that lie entirely between the amplification primers, while still maintaining the specificity derived from use of an internal probe. Overlap between the hybridization regions of the amplification and signal primers allows for flexibility in assay design and a reduction in overall amplicon length, with the resulting potential for enhanced amplification efficiency. This is important because flexibility in system design is necessary to avoid primer:primer interactions, restriction enzyme recognition sites, amplicon secondary structure and regions of excessively high G-C content. Overlap of the signal primer and an amplification primer may also enhance allelic discrimination by providing competition between closely related sequences for hybridization to the target sequence. In such a system containing two signal primers, one specific for each of two alleles at a given locus, hybridization of the specific signal primer is thermodynamically favored but formation of this structure is the result of competition for hybridization to the target of both the amplification primer and the mismatched signal primer.

An advantage of the preferred embodiments of the disclosed methods is the ability to detect sequence variations in a broad range of clinical samples without the need for extensive sample processing. The disclosed methods for detection of SNPs using specific signal primers in conjunction with SDA offer the ability to perform genotyping with a variety of sample types including blood, urine and buccal swabs without prior purification of nucleic acid. The lack of a significant sample processing required greatly reduces cost and provides improved turnaround time for results.

The signal primer adapter-mediated universal detection system of the invention provides a simple, rapid, sensitive and specific method for SNP analysis, haplotyping and detection of other nucleotide acid sequence variations. The most preferred embodiment of the invention involves homogeneous real-time genotyping of a sample including forensic samples such as blood, tissue and body fluid samples using SDA with minimal sample processing. The present invention is a powerful tool for genotyping in clinical diagnostics, forensics and drug discovery with or without nucleic acid sample preparation.