A wide variety of techniques for amplifying nucleic acids are known in the art, including, but not limited to, PCR (polymerase chain reaction), rolling circle amplification, and transcription mediated amplification. (See, e.g., Hatch et al. (1999) “Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection” Genet Anal. 15:35-40; Baner et al. (1998) “Signal amplification of padlock probes by rolling circle replication” Nucleic Acids Res. 26:5073-8; and Nallur et al. (2001) “Signal amplification by rolling circle amplification on DNA microarrays” Nucleic Acids Res. 29:E118.) A labeled primer and/or labeled nucleotides are optionally incorporated during amplification. In many embodiments, the nucleic acids of interest are captured and amplified or detected but without an ability distinguish between two or more nucleic acids from the same sample. Further, the enzyme dependent amplification techniques often run inconsistently depending on the purity and complexity of the samples provided.
PCR amplifications are commonly used in nucleic acid analysis of samples, but suffer from limited amplicon size and difficulties providing conditions for consistent enzymatic activity. These problems are only heightened in analyses requiring reliable quantitation. For example, performance of quantitative PCR (QPCR) has faired poorly in quantitation of complex or degraded samples because it is generally limited to 75-85 by amplicon size, and multiple pooled gene-specific primers are required. QPCR requires a much greater nucleic acid purity than the bDNA assay and thus more steps to process the samples prior to analysis compared to the bDNA technology. A second problem that affects RNA quantification by QPCR is the required reverse transcription step to concert mRNA sequences of interest to cDNA. This enzymatic reaction is impeded by any base modifications, by secondary mRNA structure and by impurities in the RNA preparation. Although, introduction of a high temperature heating step during PCR amplification steps may partially reverse some of the RNA base modifications, for many samples these modifications are irreversible. Older samples are often so impaired that a decrease in average QPCR signal is >90%, requiring more input RNA and increasing Ct values to 35-40 (Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K: Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples, Nucleic Acids Res 1999, 27:4436-4443). With all these problems, QPCR has not been a satisfactory method of quantitating many types of nucleic acid samples. Furthermore these problems can not be cured by continued PCR amplification of the sample. Input of PCR products of a first amplification into a second full series of PCR amplifications only tends to further compound the amplification of errors originating in the first amplification.
One method of DNA amplification has the distinct advantage of not being dependent on enzymes to generate a signal. In a typical bDNA assay for gene expression analysis, a target mRNA whose expression is to be detected is released from cells and captured by a capture probe (CP) on a solid surface (e.g., a well of a microtiter plate) through synthetic oligonucleotide probes called capture extenders (CEs). Each capture extender has a first polynucleotide sequence that can hybridize to the target mRNA and a second polynucleotide sequence that can hybridize to the capture probe. Typically, two or more capture extenders are used. Probes of another type, called label extenders (LEs), hybridize to different sequences on the target mRNA and to sequences on an amplification multimer. Additionally, blocking probes (BPs), which hybridize to regions of the target mRNA not occupied by CEs or LEs, are often used to reduce non-specific target/probe binding. A probe set for a given mRNA thus consists of CEs, LEs, and optionally BPs for the target mRNA. The CEs, LEs, and BPs are complementary to nonoverlapping sequences in the target mRNA, and are typically, but not necessarily, contiguous. Signal amplification begins with the binding of the LEs to the target mRNA. An amplification multimer is then typically hybridized to the LEs. The amplification multimer has multiple copies of a sequence that is complementary to a label probe (it is worth noting that the amplification multimer is typically, but not necessarily, a branched-chain nucleic acid; for example, the amplification multimer can be a branched, forked, or comb-like nucleic acid or a linear nucleic acid). A label, for example, alkaline phosphatase, is covalently attached to each label probe. (Alternatively, the label can be non-covalently bound to the label probes.) In the final step, labeled complexes are detected, e.g., by the alkaline phosphatase-mediated degradation of a chemilumigenic substrate, e.g., dioxetane. Luminescence is reported as relative light unit (RLUs) on a microplate reader. The amount of chemiluminescence is proportional to the level of mRNA expressed from the target gene.
An exemplary embodiment of bDNA technology is schematically illustrated in FIG. 1, wherein a single target nucleic acid is captured and detected as an accumulation of label probes. A cell or tissue sample is lysed to produce a lysate including target nucleic acid 114. The target nucleic acid 114 (e.g., an mRNA whose expression is to be detected) is captured by capture probe 104 on solid support 101 (e.g., a well of a microtiter plate) through set 111 of synthetic oligonucleotide capture extenders. Each capture extender has a first polynucleotide sequence C-3 (152) that can hybridize to the target nucleic acid and second polynucleotide sequence C-1 (151) that can hybridize to the capture probe through sequence C-2 (150) in the capture probe. Typically, two or more capture extenders are used; optionally, one CE can be used to capture a target. Each label extender in label extenders set 121 hybridizes to a different sequence on the target nucleic acid, through sequence L-1 (154) that is complementary to the target nucleic acid, and to sequence M-1 (157) on amplification multimer (141), through sequence L-2 (155). Blocking probes (124), which hybridize to sequences in the target nucleic acid not bound by either capture extenders or label extenders, are often used in bDNA assays to reduce non-specific target probe binding. A probe set for a given target nucleic acid thus consists of capture extenders, label extenders, and optional blocking probes 124 for the target nucleic acid. The capture extenders, label extenders, and optional blocking probes are complementary to non-overlapping sequences in the target nucleic acid, and are typically, but not necessarily, contiguous. In this example, a single blocking probe is used; typically, an array of different blocking probes is used in an optimized bDNA assay.
Signal amplification can begin with the binding of the label extenders to the target nucleic acid. The amplification multimer is then hybridized to the label extenders. The amplification multimer has multiple copies of sequence M-2 (158) that is complementary to label probe 142. Label 143, for example, a fluorescent group, is covalently attached to each label probe. In the final step, labeled complexes are detected, e.g., by fluorometry. The amount of fluorescence can be proportional to the level of target nucleic acid originally present in the sample (a relationship describable, e.g., by a regression curve). However, the amplifications of a bDNA assay are limited, e.g., by the number of label probe sequence sites available on the amplification multimer and stearic hindrance in the amplification complex. When detecting two or more different target nucleic acids from the same sample, the typical bDNA assay provides only a combined result without separate identification or quantitation.
In view of the above, a need exists for methods of amplifying nucleic acids to a higher degree. It would be desirable to have systems that can highly amplify signals associated with the presence of a particular nucleic acid of interest in a sample without the use of amplifying enzymes. Benefits can be obtained from methods and systems capable of amplifying, quantitating and uniquely identifying signals from two or more different nucleic acid targets in the same assay. The present invention provides these and other features that will be apparent upon review of the following.
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