WO2005110596A2 - Methods of quantifying targets in multiple samples - Google Patents

Abstract

Methods and compositions for relative abundance quantitative PCR (RT-Q-PCR) are provided. Kits and systems for practicing the methods are also provided.

Description

METHODS OF QUANTIFYING TARGETS IN MULTIPLE SAMPLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. provisional patent application USSN

60/569,822 filed May 10, 2004. The subject application claims priority to and benefit of this application, which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] This invention is in the field of quantitative PCR (Q-PCR). The invention can be used for both real time and end-point Q-PCR and can be used for detection of multiple Q-PCR amplification products in a single multiplex reaction.

BACKGROUND OF THE INVENTION

[0003] RNA production is central to all of biology. As has been understood for roughly half a century, messenger RNA (mRNA) is translated in the cell into proteins, which carry out most cellular operations. For example, in eukaryotes, mRNA is typically produced from nuclear RNA (nRNA), which is an RNA copy of a region of genomic DNA, by various splicing mechanisms. RNAs in general are typically encoded by genomic DNAs (gDNAs), with either mRNA or nRNA being produced by transcription of such DNA. This paradigm of DNA to RNA to protein is sometimes referred to as the "central paradigm" of molecular biology. Despite a few variations, such as those practiced by various RNA viruses (which can, e.g., have an RNA genome that is reverse transcribed into DNA and then replicated by transcription of the DNA back into RNA), this paradigm describes a basic way in which organisms encode traits. See also, Alberts et al. (2002) Molecular Biology of the Cell 4^th Edition Taylor and Francis, Inc., ISBN: 0815332181 ("Alberts"), and Lodish et al. (1999) Molecular Cell Biology, 4^th Edition W H Freeman & Co, ISBN: 071673706X ("Lodish").

[0004] Accordingly, detection of RNA types and levels of expression provide a basic tool for molecular biology and molecular medicine. For example, somatic or germlme polymorphisms and/or mutations can be identified by detecting the polymorphism or mutation in RNA derived from a relevant individual (e.g., from a tissue or cell of the individual). The type or level of RNA expression in a cell or tissue can also be diagnostic of disease, indicative of gene copy number, or, e.g., an indicator for the cell or tissue type that the RNA is expressed in. The detection of RNAs from pathogens (viruses such as HIV, plasmodium, bacterium and the like) and their copy number, is also diagnostic for and prognostic of disease. See also, Alberts and Lodish, id.

[0005] Because there is a direct correlation between RNA expression and cellular and organismal function, a number of methods have been developed for detecting RNAs of interest from a relevant biological sample. These methods all face various difficulties, derived, in part, from problems surrounding RNA manipulations generally. For example, enzymes that degrade RNAs are ubiquitous in the environment, causing degradation of RNA samples. Similarly, chemicals used to inhibit RNAse enzymes actually modify the RNA, making it unsuitable for certain further processing steps (e.g., reverse transcription and cloning). These difficulties make quantification of RNAs in a biological sample particularly problematic.

[0006] Rather than simply performing a northern blot for direct detection of RNA in cells, various reverse transcription/ amplification methods (that can, but usually do not, include hybridization methods such as northern blotting) are commonly used for detection of all but the most abundant RNAs, in an effort to overcome the instability of RNA during laboratory manipulations and to amplify the number of copies of RNA to be detected (thereby increasing RNA probe signal in a relevant assay). For example, reverse transcription/ amplification detection approaches, including those that rely on RT-PCR, T₇ RNA polymerase-mediated transcription/amplification, the Van Gelder Eberwine reaction, Qβ replicase amplification, and others are in common use.

[0007] During the polymerase chain reaction (PCR), target nucleic acids comprising sequences complementary to amplification primers are amplified exponentially. Quantitative PCR (Q-PCR) can be employed to determine the relative abundance of target template (e.g., a reverse-transcribed cDNA derived by reverse transcription from an RNA in a biological sample), using a control template containing a known quantity as a reference for quantification. Q-PCR is useful for quantifying RNAs that are present in an initial reverse transcription reaction used to provide the target nucleic acid for PCR. Because the cycle thresholds for both the target and reference templates during Q-PCR are measured during the logarithmic phase of amplification, before the amplification product accumulation begins to plateau in the later amplification cycles, Q-PCR reactions are typically monitored in real-time, e.g., using endonuclease probes ("TaqMan™") or molecular beacons. Therefore, current Q-PCR typically requires both a camera and specialized software to record and plot fluorescence values at each cycle during amplification, in order to determine the point at which the signal-to-background ratio becomes statistically significant, and to compare target product curves to control curves to quantify the targets. Furtheπnore, capturing a particular time slice of a PCR for comparison of signal from a target nucleic acid to a control for quantification is inherently problematic. [0008] In co-amplification PCR, end-point quantification is achieved by spiking a known amount of a standard template which shares the same amplification priming sites with the target sequence of unknown abundance, but which can differ in sequence between the two priming sites (i.e., in the resulting amplicon). Under such conditions, amplification of the spiked standard and unknown occur simultaneously in solution using the same set of primers, and the relative quantification of the unknown target concentration is determined from the amount of standard that is required to generate an equivalent amplification product. Typically, the standard and target differ in length, and are distinguished based on size separation, for instance, on agarose gels. However, co-amplification can also be carried out when the standard and target templates are so similar in sequence that they are capable of hybridizing to each other during amplification. In these reactions, the relative abundances of the species are distinguished based on subtle sequence differences that are detected by differentially labeled (e.g. by fluorescence) sequence-specific probes, such as molecular beacons. See also, US Patent 6,461,817.

[0009] End-point reaction comparisons, which are inherently simpler than real time quantification methods, are nevertheless problematic for product quantification by Q-PCR by current methods. The present invention overcomes several limitations of current Q-PCR methods, providing both real-time and end-point Q-PCR. These and other advantages will be apparent upon review of the following.

SUMMARY OF THE INVENTION

[0010] Nucleic acid oligomers are used to tag target nucleic acids (e.g., RNA transcribed from a gene) with probe annealing sites, providing a method to quantify the expression of any transcript by either real time or end-point PCR. These methods can be used to determine the relative abundance of any nucleic acid from any two (or more) nucleic acid samples for which nucleic acid abundance is to be determined. Accordingly, methods of quantifying nucleic acids, as well as related nucleic acids and compositions are provided. Systems and kits comprising the compositions are also provided.

[0011] In a first aspect, a method of quantifying an amplified nucleic acid includes the following: (a), reverse transcribing a first nucleic acid in a first reverse transcription reaction comprising a first reverse transcription oligonucleotide comprising a first identification sequence, thereby producing a first cDNA comprising the first identification sequence; (b). reverse transcribing a second nucleic acid in a second reverse transcription reaction comprising a second reverse transcription oligonucleotide comprising a second identification sequence, thereby producing a second cDNA comprising the second identification sequence; (c). combining the first and second cDNAs in a reaction mixture; (d.) amplifying the resulting combined first and second cDNAs in the reaction mixture, thereby producing first and second amplified nucleic acids; and, (e.) detecting the first and second identification sequences in the first and second amplified nucleic acids, wherein the relative quantity of the first and second identification sequences in the amplified mixture provides an indication of the quantity of the first or second nucleic acid.

[0012] The first or second nucleic acid can be, e.g., a messenger RNA, a pathogenic

RNA, a viral RNA, or the like. The first nucleic acid can be a quantified control nucleic acid (e.g., from a sample having a standardized concentration of the control nucleic acid). The abundance of the second nucleic acid in a sample can be a diagnostic marker for one or more gene copy number, infection, disease, condition or the like, e.g., of an RNA from a biological sample.

[0013] Amplifying the first and second cDNAs optionally includes performing a

PCR reaction with a polymerase, the first and second cDNAs, a first amplification primer complementary to a portion of the first and second reverse transcription oligonucleotides, a second amplification primer complementary to the first cDNA and a third amplification primer complementary to the second cDNA, wherein the third amplification primer is the same or different than the second amplification primer. Detection of the first and second identification sequences optionally comprises detecting the first or second amplification sequences, or one or more complementary sequences thereof. Detection of the first and second identification sequences optionally comprises detecting a first probe that specifically hybridizes to the first identification sequence or a complement thereof, and detecting a second probe that specifically hybridizes to the second identification sequence or a complement thereof.

[0014] The methods can include normalizing a signal detected from the first or second probe to account for one or more difference in activity between the first or second probe. These differences can include e.g., (i.) a difference in probe labels that label the first and second probes, (ii.) a difference in amplification efficiency caused by the presence of the first or second identification sequences or complementary sequences thereof in the first or second cDNAs, (iii.) a difference in reverse transcription efficiency caused by the presence of the first or second identification sequences in the first or second reverse transcription oligonucleotides, and/or the like.

[0015] In one preferred aspect, the detection includes end point quantification of one or more probe signals from one or more probes that bind to the first or second identification sequences or complementary sequences thereof. The ability to perform quantification with an end point assay provides substantial benefits over prior art Q-PCR methods.

[0016] Optionally, the detection can include end point or real time quantification of one or more probe signals from one or more probes that bind to the first or second identification sequences or complementary sequences thereof. The detection step can comprise detecting an optical label on the one or more probes, detecting a fluorescent label on the one or more probes, and/or detecting a luminescent label on the one or more probes.

[0017] In one additional aspect, the methods optionally further include: (f). reverse transcribing the first nucleic acid in a third reverse transcription reaction comprising a third reverse transcription oligonucleotide comprising the second identification sequence, thereby producing a third cDNA comprising the first identification sequence; (g). reverse transcribing a fourth nucleic acid in a fourth reverse transcription reaction comprising a fourth reverse transcription oligonucleotide comprising the first identification sequence, thereby producing a fourth cDNA comprising the first identification sequence; and, (h). comparing an amount of first, second, third, and/or fourth cDNAs, thereby monitoring or controlling for an effect of the first or second identification sequence on the reverse transcription. Optionally, the method can also include: (i). combining the third and fourth cDNAs in a reaction mixture; (j). amplifying the resulting combined third and fourth cDNAs in the reaction mixture, thereby producing third and fourth amplified nucleic acids; and, (k). detecting an amount of the first, second, third and first amplified nucleic acids, thereby monitoring or controlling for an effect of the first and second identification sequences on amplification efficiency.

[0018] Compositions, useful, e.g., in practicing the methods herein, area also a feature of the invention. In one aspect, the composition includes a first cDNA comprising: a first target sequence complementary to a first RNA sequence of a first RNA isolated from a biological sample, a first amplification primer binding site, and a first identification sequence between the first amplification primer binding site and the first target sequence; wherein the first identification sequence comprises one or more bases that are non- complementary to the first RNA. The composition also includes a second cDNA that differs in sequence as compared to the first cDNA, the second cDNA comprising the first amplification primer binding site and a second identification sequence proximal to the first amplification primer binding site, wherein the first and second identification sequences are distinguishable.

[0019] The first identification sequence can be complementary to a molecular beacon or an endonuclease probe used in quantification of amplification products of the compositions. As noted above, the first RNA sequence can be any relevant RNA, e.g., an mRNA, a pathogenic RNA sequence, a viral RNA sequence, or the like. The second cDNA can include a control sequence complementary to a second RNA sequence or an RNA isolated from a biological sample. The control sequence can be the same as the first target sequence, or different than the first target sequence. Typically, the second identification sequence is between the control DNA sequence and the first amplification primer binding site.

[0020] The composition optionally includes additional elements, e.g., depending on the use contemplated for the composition. For example, where the composition is an amplification reaction, the composition can include, e.g., a polymerase, an amplification primer complementary to the first introduced amplification primer binding site, a dNTP, a first detection probe that specifically hybridizes to the first identification sequence a second detection probe that hybridizes to the second identification sequence, and/or the like.

[0021] The composition is optionally packaged into a kit. The kit can include the composition, or elements of the composition, contained in one or more containers. The kit optionally also includes appropriate packaging materials and instructions in use of the kit. [0022] In one aspect, a reverse transcription primer is provided. This primer includes, for example, a primer that includes a first 3' oligonucleotide subsequence that is complementary to a first subsequence of an RNA isolated from a biological sample. The primer also includes a middle oligonucleotide subsequence complementary to a selected probe sequence, which probe sequence is different than a proximal sequence of the RNA located adjacent to the first subsequence. A 5' oligonucleotide sequence that is complementary to the RNA is also provided. This primer can be present, e.g., in a reverse transcription reaction composition used to produce cDNA in the composition noted above. A kit comprising the RNA primer is, optionally, a feature of the invention. The kit can further include, e.g., a container that contains the primer, one or more reverse transcription reagents, packaging materials and instructions in using the kit components.

[0023] In a further aspect, a system comprising a pair of reverse transcription reaction mixtures is provided. The system includes a first RNA and a first transcriptase- extendible oligonucleotide comprising a first transcriptase-extendible subsequence complementary to the first RNA, a first identification sequence, and a first amplification primer binding site, wherein the first identification sequence comprises one or more nucleotides that are non-complementary to the first RNA, and wherein the first identification sequence is between the amplification primer binding site and the extendible subsequence. The system additionally includes a second reaction mixture comprising a second RNA that is different than the first RNA and a second transcriptase-extendible oligonucleotide partially complementary to the second RNA, the second transcriptase- extendible oligonucleotide comprising a second extendible subsequence complementary to the second RNA, a second identification sequence that is different from the first identification sequence, wherein the second identification sequence comprises one or more nucleotides that are non-complementary to the first or second RNA, and wherein the second identification sequence is between the first amplification primer binding site and the second extendible subsequence. The first and second second identification sequences typically include, or are complementary to, a molecular beacon or an endonuclease probe sequence. The system optionally further includes detection elements (microscopes, PMTs, photodiodes, cameras, CCDs, or the like) and/or fluid handling elements for forming the reaction mixtures or dispensing products of the reactions. These can be, e.g., classical fluid handling elements (e.g., pipettors), high throughput fluid handlers (e.g., robotic systems) or microfiuidic devices. Kits for forming the system of the invention, e.g., comprising one or more components oi me reaction mixtures, in appropriate containers and packaging materials, and optionally including instructional materials, are also a feature of the invention.

DEFINITIONS

[0024] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein, h describing and claiming the present invention, the following terminology can be used in accordance with the definitions set out below. [0026] A "nucleic acid" encompasses any physical string of monomer units that can be corresponded to a sequence of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA in solution, such as 2'-O- methylated oligonucleotides), and the like. A nucleic acid can be e.g., single-stranded or double-stranded. Unless context dictates otherwise, an indicated nucleic acid strand optionally includes the complementary strand thereof.

[0027] A "nucleotide sequence" is a polymer of nucleotides (an oligonucleotide, a

DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified nucleotide sequence (e.g., through application of standard Watson-Crick base pairing rules).

[0028] An "oligonucleotide" is a polymer comprising two or more nucleotides. The polymer can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The nucleotides of the oligonucleotide can be natural or non- natural and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like.

[0029] A "primer" is a nucleic acid that contains a sequence complementary to a region of a template nucleic acid strand that primes the synthesis of a strand complementary to the template (or a portion thereof). In the context of the invention, primers are typically, but need not be, chemically synthesized oligonucleotides. hi an amplification, e.g., a PCR amplification, a pair of primers typically define the 5' ends of the two complementary strands of the nucleic acid target that is amplified. In order to be extendable by a standard polymerase, a primer typically has a free 3' hydroxyl group.

[0030] A "cDNA" is a DNA made using an RNA template.

[0031] A nucleic acid is "amplified" when one or more copies of the nucleic acid, or at least one strand thereof, are copied and/or transcribed. Thus, a DNA can be amplified to produce DNAs or RNAs, and RNA can be amplified by transcribing it from DNA (which, itself, can be made by reverse transcription of an RNA), etc.

[0032] An "amplicon" is a molecule made by copying or transcribing another molecule, e.g., as occurs in PCR, transcription, and/or cloning. [0033] A "template nucleic acid" is a nucleic acid that is to be copied or transcribed.

[0034] An "end point" quantification of material in a reaction mixture is performed when the production of new product by the reaction mixture has essentially stopped. For example, a PCR endpoint occurs when either (1) the PCR runs out of one or more necessary 0 reagent (e.g., active polymerase, nucleotide, etc.), or (2) when the reaction is stopped, hi general, reactions can be stopped by any of a variety of mechanisms, depending on context, e.g., by ceasing cycles of amplification in a PCR, or by adding one or more enzymatic inhibitors such as EDTA to reduce or eliminate enzymatic activity in the relevant reaction (PCR, reverse transcription, or the like), or by heating or cooling the reaction mixture.

[0035] A "biological sample" is a sample isolated from or derived from a biological source, such as a cell, cell culture, tissue, biological fluid (e.g., blood, urine, etc.), biological waste, or the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic illustration of a method of tagging cDNAs for RA-Q-

PCR. hi panels A and D, mRNA from a specific gene is shown as wavy lines, and is present in different amounts in this example. Reverse transcription with two different oligomers that contain the same priming sequence is used to incorporate two different sequences (e.g., molecular beacon annealing sites) into cDNAs.

[0037] Figure 2 is a schematic illustration of PCR amplification reactions performed on tagged cDNAs produced as in Figure 1. Once the cDNAs are tagged, the samples are combined in a single tube, and PCR amplification is carried out using primers, shown as forward and reverse arrows, which flank the molecular beacon annealing sites, as shown.

[0038] Figure 3 is a schematic overview of a reverse transcription tagging and amplification strategy along with corresponding schematic data charts showing signals from the amplification reaction.

DETAILED DESCRIPTION [0039] Methods and related compositions for the detection of relative amounts of specific sequences in two or more samples in a single real time or end-point amplification assay are provided. The assay is referred to herein as a "relative abundance Q-PCR" (RA- Q-PCR). The methods and related compositions described herein provide ways of quantifying relative abundance of target DNAs in multiple samples without the disadvantages of traditional Q-PCR, extending the capabilities of Q-PCR co-amplification methods.

[0040] It is well established that, under conditions in which the spiked standard and an unknown differ in initial concentration in a PCR, that a linear relationship exists between this relative initial difference and the relative abundances of resulting amplicons at any stage of the PCR, including the end-point. With RA-Q-PCR, a linear relationship also exists between an initial template molar ratio of standard to target and the molar ratio of products at subsequent cycles, including the end-point. RA-Q-PCR augments current co- amplification methods to provide a determination of the relative abundance of any target from any two (or more) nucleic acid samples to be determined. An example to illustrate such a use is the relative quantification of the abundance of a given RNA sequence in two different samples. The method involves "tagging" these sequences in an initial step with an identification sequence. This tagging step can include a linear or exponential conversion of the RNA into (for example) a quantitative single-stranded or double-stranded cDNA representation. This conversion of RNA into cDNA is preferably performed by reverse transcription. Alternatively, the conversion can involve amplification of a single-stranded cDNAbyPCR.

[0041] Aspects of this first conversion step are that 1) these conversions are achieved for the two samples independently, i.e., the samples are not pooled during conversion; 2) these conversions occur with reasonably similar efficiency for both samples independently, such that the relative change in abundance resulting from the conversion is reasonably the same in each sample; and 3) the resulting sequences are altered in such a way that the two samples can be distinguished from each other, such as by length analysis or hybridization to sequence-specific probes.

[0042] The method then involves a second step, in which the two samples are combined, and amplified with a single set of primers which anneal identically to the target region of both samples (in other words, the priming sequences do not differ between the two samples). Under such conditions, co-amplification occurs, and the relative abundances of the sequences can be measured either in real-time or as an end point analysis. Although typically described in terms of polymerase-mediated amplification below for convenience, it will be appreciated that ligase-mediated amplification, or amplification by transcription can be similarly utilized in the methods herein.

[0043] Length analysis or hybridization of a probe (molecular beacon, TaqMan™ probe, etc.) to the identification sequence tag is performed in a quantitative fashion, providing a measure of the relative abundance of the amplicons corresponding to the different samples. If one of the samples has a known initial concentration, then the concentration of the second sample is determined from comparison to the known sample.

OVERVIEW

[0044] A method of tagging cDNAs for RA-Q-PCR is schematically illustrated in

Figure 1. In panels A and D, mRNA from a specific gene is shown as wavy lines, and is present in different amounts in this example. Reverse transcription with two different oligomers that contain the same priming sequence is used to incorporate two different identification sequences (e.g., molecular beacon annealing sites in this instance, but the identification can be any distinguishable probe binding site, or size differentiation sequence, or restriction enzyme binding site, or other site that can be differentiated in a downstream detection assay;, snown as solid and dot-dash-dot loops (panels B and E), into cDNA samples labeled A and B (panels C and F). The incorporation of entire molecular beacon annealing sites into cDNAs is schematically shown for purposes of illustration, but any alteration that allows the differential detection of cDNAs in a probe based or length-based assay can be used.

[0045] In addition to tagging modifications during reverse transcription, cDNAs can also be tagged with modified amplification primers during PCR, or by incorporating different nucleotides (for instance, 5-me-C) into the amplification products. Once the cDNAs are tagged, the samples are combined in a single tube, and PCR amplification is carried out using primers, shown as forward and reverse arrows, which flank the molecular beacon annealing sites (Figure 2). In the schematic illustration, at the end of PCR, the more abundant tagged target contains a FAM fluorophore, while the less abundant tagged target contains a HEX fluorophore. Figure 3 also schematically illustrates this reverse transcription and Q-PCR, with two different incorporation sites (solid boxes on the reference sample, open boxes on the test sample). Figure 3 additionally shows schematic data from the amplification, illustrating the signal differences that are provided by the overall procedure. In the schematic graphs, the x axis shows time and the y axis shows signal intensity from binding of a probe to the identification sequences. [0046] The methods described here for quantification of targets have several advantages over existing technology. Targets from multiple samples can be quantified using an end-point assay rather than Q-PCR, assays require a single tube, a plate reader can be used for detection, results are independent of primer design, results are independent of transcript abundance, and results are independent of amplification efficiency.

[0047] An additional detailed illustrative example (shown for detection of SOD genes) is described below using a dual labeled probe.

TARGET NUCLEIC ACIDS

[0048] The target nucleic acids of interest that are to be reverse transcribed, amplified and detected in the methods of the invention can be essentially any nucleic acid(s). The sequences for many nucleic acids and amino acids (from which nucleic acid sequences can be derived via reverse translation) are available. No attempt is made to identify the millions of known nucleic acids, any of which can be detected in the methods of the invention. Common sequence repositories for known nucleic acids include GenBank® fiMBL, DDB J and the NCBϊ. Other repositories can easily be identified by searching the internet. The initial form of the target nucleic acid can be an RNA (e.g., where amplification includes RT-PCR or RT-LCR, the Van-Gelder Eberwine reaction or Ribo- SPIA) or can initially be DNA (e.g., cDNA or genomic DNA) that is transcribed to form RNA that is then reverse transcribed to form cDNA. In an alternate embodiment, the nucleic acid is simply a DNA molecule and the tagging strategy described herein is achieved using amplification primers that comprise identification tag sequences.

[0049] In general, the nucleic acid can be any analogue of RNA or DNA (e.g., for detection of synthetic nucleic acids or analogues thereof, where the sample of interest includes artificial nucleic acids). Any variation in a nucleic acid can be detected, e.g., a mutation, a polymorphism, a single nucleotide polymorphism (SNP), an allele, an isotype, a haplotype, etc. Further, because the present invention provides for quantification of the nucleic acid, variation in expression levels or gene copy numbers can be detected by the methods herein. Common targets for analysis include those that are relevant to disease, e.g., mRNA, viral RNA, pathogenic RNA, etc.

[0050] For example, the methods of the invention are useful in screening samples derived from patients for a nucleic acid of interest, e.g., from bodily fluids (blood, urine etc.), tissue, and/or waste from the patient. Thus, stool, sputum, saliva, blood, lymph, tears, sweat, urine, vaginal secretions, ejaculatory fluid or the like can easily be screened for nucleic acids by the methods of the invention, as can essentially any tissue of interest. These samples are typically taken, following informed consent, from a patient by standard medical laboratory methods.

[0051] Prior to reverse transcription and amplification, nucleic acids are optionally purified from the samples by any available method, e.g., those taught in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al, Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001 ("Sambrook"); and/or Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) ("Ausubel")). A plethora of kits are also commercially available for the purification of nucleic acids from cells or other samples (see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean^1M, rrom tratagene; ana, QIAprep™ from Qiagen). Alternately, samples can be directly subjected to reverse transcription and amplification, e.g., following aliquotting and any appropriate dilutions.

[0052] One class of nucleic acids of interest to be detected in the methods herein are those involved in cancer. Any nucleic acid that is associated with cancer can be detected in the methods of the invention, e.g., those that encode over expressed or mutated polypeptide growth factors (e.g., sis), overexpressed or mutated growth factor receptors (e.g., erb-Bl), over expressed or mutated signal transduction proteins such as G-proteins (e.g., Ras), or non-receptor tyrosine kinases (e.g., abl), or over expressed or mutated regulatory proteins (e.g., myc, myb, jun, fos, etc.) and/or the like. In general, cancer can often be linked to signal transduction molecules and corresponding oncogene products, e.g., nucleic acids encoding Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and/or nuclear receptors. p53, colloquially referred to as the "molecular policeman" of the cell, is of particular relevance, as about 50% of all known cancers can be traced to one or more genetic lesion in p53.

[0053] Taking one class of genes that are relevant to cancer as an example for discussion, many nuclear hormone receptors have been described in detail and the mechanisms by which these receptors can be modified to confer oncogenic activity have been worked out. For example, the physiological and molecular basis of thyroid hormone action is reviewed in Yen (2001) "Physiological and Molecular Basis of Thyroid Hormone Action" Physiological Reviews 81(3): 1097-1142, and the references cited therein. Known and well characterized nuclear receptors include those for glucocorticoids (GRs), androgens (ARs), mineralocorticoids (MRs), progestins (PRs), estrogens (ERs), thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs), and the peroxisome proliferator activated receptors (PPARs) that bind eicosanoids. The so called "orphan nuclear receptors" are also part of the nuclear receptor superfamily, and are structurally homologous to classic nuclear receptors, such as steroid and thyroid receptors. Nucleic acids that encode any of these receptors, or oncogenic forms thereof, can be detected in the methods of the invention. About 40% of all pharmaceutical treatments currently available are agonists or antagonists of nuclear receptors and/or oncogneic forms thereof, underscoring the relative importance of these receptors (and their coding nucleic acids) as targets for analysis by the methods of the invention. [0054] Another example class of nucleic acids of interest are those that are diagnostic of colon cancer. Colon cancer is a common disease that can be sporadic or inherited. The molecular basis of various patterns of colon cancer is known in some detail. In general, germline mutations are the basis of inherited colon cancer syndromes, while an accumulation of somatic mutations is the basis of sporadic colon cancer. In Ashkenazi Jews, a mutation that was previously thought to be a polymorphism may cause familial colon cancer. Mutations of at least three different classes of genes have been described in colon cancer etiology: oncogenes, suppressor genes, and mismatch repair genes. One example nucleic acid encodes DCC (deleted in colon cancer), a cell adhesion molecule with homology to fibronectin. An additional form of colon cancer is an autosomal dominant gene, hMSH2, that comprises a lesion. Familial adenomatous polyposis is another form of colon cancer with a lesion in the MCC locus on chromosome #5. For additional details on Colon Cancer, see, Calvert et al. (2002) "The Genetics of Colorectal Cancer" Annals of Internal Medicine 137 (7): 603-612 and the references cited therein. For a variety of colon cancers and colon cancer markers that can be detected, see, e.g., Boland (2002) "Advances in Colorectal Cancer Screening: Molecular Basis for Stool-Based DNA Tests for Colorectal Cancer: A Primer for Clinicans" Reviews In Gastroenterological Disorders Volume 2, Supp. 1 and the references cited therein. Biological samples derived from patient tissue or even stool can be used to detect types and levels of expression of relevant nucleic acids, using the methods herein.

[0055] Cervical cancer is another example of a preferred target for detection, e.g., in samples obtained from vaginal secretions. Cervical cancer can be caused by the papova virus and has two oncogenes, E6 and E7. E6 binds to and removes p53 and E7 binds to and removes PRB. The loss of p53 and uncontrolled action of E2F/DP growth factors without the regulation of pRB is one mechanism that leads to cervical cancer.

[0056] Another preferred target for detection by the methods of the invention is retinoblastoma, e.g., in samples derived from tears. Retinoblastoma is a tumor of the eyes which results from inactivation of the pRB gene. It has been found to transmit heritably when a parent has a mutated pRB gene (and, of course, somatic mutation can cause non- heritable forms of the cancer).

[0057] Neurofibromatosis Type 1 can be detected in the methods of the invention.

The NF1 gene is inactivated, which activates the GTPase activity of the ras oncogene. If NF1 is missing, ras is overactive ana causes neural tumors. The methods of the invention can be used to detect Neurofibromatosis Type 1 in CSF or via tissue sampling.

[0058] Many other forms of cancer are known and can be found by detecting associated genetic lesions using the methods of the invention. Cancers that can be detected by detecting appropriate lesions include cancers of the lymph, blood, stomach, gut, colon, testicles, pancreas, bladder, cervix, uterus, skin, and essentially all others for which a known genetic lesion exists. For a review of the topic, see, The Molecular Basis of Human Cancer Coleman and Tsongalis (Eds) Humana Press; ISBN: 0896036340; 1st edition (August 2001).

[0059] Similarly, nucleic acids from pathogenic or infectious organisms can be detected by the methods of the invention, e.g., for infectious fungi, e.g., Aspergillus, or Candida species; bacteria, particularly E. coli, which serves a model for pathogenic bacteria (and, of course certain strains of which are pathogenic), as well as medically important bacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodid), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as ( + ) RNA viruses (examples include Poxviruses e.g., vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), ( - ) RNA viruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.

[0060] A variety of nucleic acid encoding enzymes (e.g., industrial enzymes) can also be detected according to the methods herein, such as amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases. Similarly, agriculturally related proteins such as insect resistance proteins (e.g., the Cry proteins), starch and lipid production enzymes, plant and insect toxins, toxin- resistance proteins, Mycotoxin detoxification proteins, plant growth enzymes (e.g., Ribulose 1,5-Bisphosphate arboxyiase/υxygenase, "RUBISCO"), lipoxygenase (LOX), and Phosphoenolpyruvate (PEP) carboxylase can also be detected.

MAKING OLIGONUCLEOTIDES

[0061] In general, synthetic methods for making oligonucleotides, including probes, molecular beacons, PNAs, LNAs (locked nucleic acids), etc., which can be used as reagents for the production and/or detection of nucleic acids, e.g., reverse transcription products and amplicons made according to the methods herein, are well known. For example, oligonucleotides can be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20): 1859-1862, e.g., using a commercially available automated synthesizer, e.g., as described in Needham-VaiiDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides, including modified oligonucleotides can also be ordered from a variety of commercial sources known to persons of skill. There are many commercial providers of oligo synthesis services, and thus this is a broadly accessible technology. Any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (www.genco.com), ExpressGen ie. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Similarly, PNAs can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc.

(www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio -Synthesis, Inc., and many others.

REVERSE TRANSCRIPTION

[0062] RT-PCR is a common procedure that results in the production of cDNA amplicons that correspond to RNAs (e.g., mRNAs) used as original template materials. In brief, the protocol includes contacting an mRNA with a reverse transcriptase, in the presence of an appropriate extendible primer as detailed herein. The reverse transcriptase copies the mRNA into a first cDNA strand, which can then be amplified according to standard PCR or LCR methods. See also, Imiis, Sambrook and Ausubel, supra.

AMPLIFICATION

[0063] Amplification methods include polymerase based methods, or, alternately, those that are ligase mediated. These include PCR or LCR, in vitro transcription, and/or the like. PCR, RT-PCR and LCR are in particularly broad use, in many different fields. Details regarding the use ot ttiese ana otner amplification methods can be found in any of a variety of standard texts, including, e.g.,: See also, Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual 3rd Edition Cold Spring Harbor Laboratory, ISBN: 0879695773 ("Sambrook"); Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, hie, (supplemented through 2003) ("Ausubel"); and PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis). Many available biology texts also have extended discussions regarding PCR and related amplification methods. Additional details regarding nucleic acid amplification can be found in Mullis et al, (1987) U.S. Patent No. 4,683,202. [0064] Sample-specific methods of performing amplification are also well known.

That is, amplification protocols and sample preparation methods can vary, depending on the sample of interest, and the literature provides considerable details in this respect. For example, details regarding amplification of nucleic acids in plants can be found, e.g., in Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific Publishers, Inc. Similarly, additional details regarding amplifications for cancer detection can be found in any of a variety of sources, e.g., Bernard and Wittwer (2002) "Real Time PCR Technology for Cancer Diagnostics Clinical Chemistry 48(8):1178-1185; Perou et al. (2000) "Molecular portraits of human breast tumours" Nature 406:747-52; van't Veer et al. (2002) "Gene expression profiling predicts clinical outcome of breast cancer" Nature 415:530-6; Rosenwald et al. (2001) "Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia" J Exp Med 194:1639-47; Alizadeh et al. (2000) "Distinct types of diffuse large B-cell ly phoma identified by gene expression profiling" Nature 403:503-11; Garber et al. (2001) "Diversity of gene expression in adenocarcinoma of the lung" Proc Natl Acad Sci U S A 98: 13784-9; Tirkkonen et al. (1998) "Molecular cytogenetics of primary breast cancer by CGH" Genes Chromosomes Cancer 21 :177-84; Watanabe et al. (2001) "A novel amplification at 17q21- 23 in ovarian cancer cell lines detected by comparative genomic hybridization" Gynecol Oncol 81:172-7, and many others.

[0065] One of skill will also appreciate that essentially any RNA can be converted into a DNA suitable for restriction digestion, PCR or LCR amplification, and/or downstream manipulations (such as sequencing or cloning), e.g., using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra. AMPLICON DETECTION

[0066] hi the present invention, different identification sites are incorporated into different reverse transcription oligonucleotides that are eventually incorporated into cDNAs, which, in turn, are amplified. The identification sites can include a sequence (or the complement thereof) that is bound by a probe such as a dual labeled probe (FRET probe, molecular beacon, endonuclease probe (TaqMan™), etc.), a conventional probe binding site, a restriction enzyme site, or any other detectable difference in sequence. The amplicon produced by amplification of the cDNA is detected in either an endpoint or real time assay, by monitoring a signal with the appropriate signal monitoring equipment, detecting the difference in sequence. For example, Sambrook provides detailed information on restriction enzyme digestion methods, which can be applied to the present invention as noted herein.

[0067] Indeed, any available method for sequence-specific detection of amplified nucleic acids can be used in the present invention. Common approaches include real time amplification detection with molecular beacons or TaqMan™ probes, detection of intercalating dyes into size-separated amplicons, detection of labels incorporated into the amplification probes or the amplified nucleic acids themselves, e.g., following electrophoretic separation of the amplification products from unincorporated label), hybridization based assays (e.g., array based assays) and/or detection of secondary reagents that bind to the nucleic acids. Details on these general approaches is found in the references cited herein, e.g., Sambrook, Ausubel, and the references in the sections herein related to real time PCR detection. Additional labeling strategies for labeling nucleic acids and corresponding detection strategies can be found, e.g., in Haugland (2003) Handbook of Fluorescent Probes and Research Chemicals Ninth Edition by Molecular Probes, Inc. (Eugene OR) (Also available on CD ROM). For illustration, two preferred technologies for detecting sequence differences are discussed in more detail below, but it will be appreciated that many others, including restriction enzyme digestion of the identification sites and electrophoresis of the products, size separation of amplification products, and the like, are also appropriate.

Molecular Beacon Probes [0068] In one preferred embodiment, molecular beacons are used to detect the identification sequences of interest. By using different fluorophores on different beacons, used to detect different identification sequences, separate signals can simultaneously be detected from a given reaction or end-point mixture. While it is expected that one of skill is fully able to use molecular beacons, a orief discussion of the technology is provided below for illustrative purposes.

[0069] Molecular beacons (MBs) are oligonucleotides designed for the detection and quantification of target nucleic acids (e.g., target DNAs). The basic principles of molecular beacon mediated target nucleic acid detection is taught, e.g., in USSN PCT/US01/13719.

[0070] As taught in the ' 719 application, the 5 ' and 3 ' termini of the MB collectively comprise a pair of moieties that confer the detectable properties of the MB. One of the termini is attached to a fluorophore and the other is attached to a quencher molecule capable of quenching a fluorescent emission of the fluorophore. For example, one example fluorophore-quencher pair can use a fluorophore such as EDANS or fluorescein, e.g., on the 5'-end and a quencher such as Dabcyl, e.g., on the 3'-end.

[0071] When the MB is present free in solution, i.e., not hybridized to a second nucleic acid, the stem of the MB is stabilized by complementary base pairing. This self- complementary pairing results in a "hairpin loop" structure for the MB in which the fluorophore and the quenching moieties are proximal to one another. In this confirmation, the fluorescent moiety is quenched by the fluorophore.

[0072] The loop of the molecular beacon is complementary to a sequence to be detected in the target nucleic acid, such that hybridization of the loop to its complementary sequence in the target forces disassociation of the stem, thereby distancing the fluorophore and quencher from each other. This results in unquenching of the fluorophore, causing an increase in fluorescence of the MB.

[0073] Further details regarding standard methods of making and using MBs are well established in the literature and MBs are available from a number of commercial reagent sources. Further details regarding methods of MB manufacture and use are found, e.g., in Leone et al. (1995) "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA." Nucleic Acids Res. 26:2150- 2155; Tyagi and Kramer (1996) "Molecular beacons: probes that fluoresce upon hybridization" Nature Biotechnology 14:303-308; Blok and Kramer (1997) "Amplifiable hybridization probes containing a molecular switch" Mol Cell Probes 11:187-194; Hsuih et al. (1997) "Novel, ligation-dependent PCR assay for detection of hepatitis C in serum" J Clin Microbiol 34:501-507; Kostrikis et al. (1998) "Molecular beacons: spectral genotyping of human alleles" Scιence2^'79^':T228-1229; Sokol et al. (1998) "Real time detection of DNA:RNA hybridization in living cells" Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998) "Multicolor molecular beacons for allele discrimination" Nature Biotechnology 16:49-53; Bonnet et al. (1999) "Thermodynamic basis of the chemical specificity of structured DNA probes" Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang et al. (1999) "Designing a novel molecular beacon for surface-immobilized DNA hybridization studies" J. Am. Chem. Soc. 121:2921-2922; Marras et al. (1999) "Multiplex detection of single-nucleotide variation using molecular beacons" Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al. (1999) "Multiplex detection of four pathogenic refroviruses using molecular beacons" Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399. Additional details regarding MB construction and use is found in the patent literature, e.g., USP 5,925,517 (July 20, 1999) to Tyagi et al. entitled "Detectably labeled dual conformation oligonucleotide probes, assays and kits;" USP 6,150,097 to Tyagi et al (November 21, 2000) entitled "Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes" and USP 6,037,130 to Tyagi et al (March 14, 2000), entitled "Wavelength-shifting probes and primers and their use in assays and kits."

[0074] MBs have wide-spread acceptance as robust reagents for detecting and quantitating nucleic acids, including in real time (MBs can be used to detect targets as they are formed). A variety of commercial suppliers produce standard and custom molecular beacons, including Cruachem (cruachem.com), Oswel Research Products Ltd. (UK; oswel.com), Research Genetics (a division of Invitrogen, Huntsville AL (resgen.com)), the Midland Certified Reagent Company (Midland, TX mcrc.com) and Gorilla Genomics, LLC (Alameda, CA). A variety of kits which utilize molecular beacons are also commercially available, such as the Sentinel™ Molecular Beacon Allelic Discrimination Kits from Stratagene (La Jolla, CA) and various kits from Eurogentec SA (Belgium, eurogentec.com) and Isogen Bioscience BV (The Netherlands, isogen.com).

Endonuclease Probes [0075] In one embodiment, an endonuclease sequence detection system such as the

"TaqMan™" system is used for detecting amplified nucleic acids. TaqMan™ operates by using the endogenous endonuclease activity of certain polymerases to cleave a quencher or label free from an oligonucleotide that comprises the quencher and label, resulting in unquenching of the label. The polymerase only cleaves the quencher or label upon initiation of replication, i.e., when the oligonucleotide is bound to the template and the polymerase extends trie primer, inus, an appropriately labeled oligonucleotide and polymerase comprising the appropriate nuclease activity can be used to detect a nucleic acid of interest. Real time PCR product analysis by, e.g., FRET or TaqMan provides well- known techniques for monitoring nucleic acid sequence that has been used in a variety of contexts, particularly for real-time PCR analysis {see, Laurendeau et al. (1999) "TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with LNK4 locus haploinsufficiency" Clin Chem 45(7):982-6; Laurendeau et al. (1999) "Quantification of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay" Clin Chem 59(12):2759-65; and Kreuzer et al. (1999) "LightCycler technology for the quantification of bcr/abl fusion transcripts" Cancer Research 59(13):3171-4. In the present invention, typical endonuclease probe methods can be used in end-point analysis, as well as in conventional real-time PCR methods.

SYSTEMS AND KITS

[0076] Systems or kits of the invention can include the reverse transcription or amplification reaction components or products described herein. Optionally, the systems will include additional components that aid in fluid handling, temperature cycling (e.g., for PCR or LCR) detection of products, or the like.

[0077] In general, the kits and systems herein optionally include signal detectors, e.g., which detect fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism or the like. Fluorescent detection is especially preferred and generally used for detection of amplified nucleic acids (however, upstream and/or downstream operations such as purification, cloning or the like can be performed on amplicons, and can involve other detection methods).

[0078] The detector(s) optionally monitors one or a plurality of signals from an amplification reaction. For example, the detector can monitor optical signals which correspond to "end point" and/or "real time" amplification assay results.

[0079] Example detectors can include any of: plate readers, photo multiplier tubes, spectrophotometers, CCD arrays, scanning detectors, microscopes, galvo-scanns and/or the like. Amplicons or other components which emit a detectable signal can be placed in proximity to the detector, or, alternatively, the detector can move relative to the site of the amplification reaction (or, the detector can simultaneously monitor a number of spatial positions corresponding to channel regions, or microtiter wells e.g., as in a CCD array). [0080]" The detector can include or be operably linked to a computer, e.g., which has software for converting detector signal information into assay result information (e.g., presence or quantity of a nucleic acid of interest), or the like.

[0081] Signals are optionally calibrated, e.g., by calibrating the system by monitoring a detectable signal from a known source.

[0082] A system can also employ multiple different detection systems for monitoring one or more signals in the system. Detection systems of the present invention are generally used to detect, quantify or otherwise monitor identification sequences in amplicons of interest.

[0083] Particularly preferred detection systems include optical detection systems for detecting an optical property of probes bound to identification sequences of amplicons. During use, such optical detection systems are typically located adjacent to the amplification reaction site of interest. Optical detection systems include systems that are capable of measuring the light emitted by the probe. In preferred aspects, the detector measures an amount of light emitted from the material, such as a fluorescent or luminescent material. As such, the detection system will typically include collection optics for gathering a light based signal and transmitting that signal to an appropriate light detector. Plate readers and/or microscope objectives of varying power, field diameter, and focal length are readily utilized as at least a portion of this optical system. The light detectors are optionally spectrophotometers, photodiodes, avalanche photodiodes, photomultiplier tubes, diode arrays, or in some cases, imaging systems, such as charged coupled devices (CCDs) and/or the like. The detection system is typically coupled to a computer, via an analog to digital or digital to analog converter, for transmitting detected light data to the computer for analysis, storage and data manipulation, e.g., to quantify one or more amplicon of interest, based upon the signals detected by the system.

[0084] In the case of fluorescent probes, the detector optionally includes a light source that produces light at an appropriate wavelength for activating the fluorescent probe, as well as optics for directing the light source to the reaction mixture of interest. The light source can be any number of light sources that provides an appropriate wavelength, including lasers, laser diodes and LEDs. Other light sources are used in other detection systems. For example, broad band light sources are typically used in light scattering/transmissivity detection schemes, and the like. Typically, light selection parameters are well known to those of skill in the art.

[0085] The detector can exist as a separate unit, but can also be integrated in the system into a single instrument. Integration of these functions into a single unit facilitates connection to a computer, by permitting the use of a few or even a single communication port(s) for transmitting information between a system controller, the detector and the computer.

[0086] The systems of the invention can include fluid manipulation devices such as pipettors, laboratory robots, microfluidic devices or the like. In addition, the systems can include detectors, sample storage elements (microtiter plates, etc.), fluid dispensors, amplification devices, computers and/or the like. These systems can be used for aliquoting, reverse transcribing amplifying and analyzing a nucleic acid of interest.

Computer [0087] As noted above, the detection system and or fluidic elements are optionally coupled to an appropriately programmed processor or computer which functions to instruct the operation of these instruments in accordance with preprogrammed or user input instructions, receive data and information from these instruments, and interpret, manipulate and report this information to the user. As such, the computer is typically appropriately coupled to one or both of these instruments (e.g., including an analog to digital or digital to analog converter as needed).

[0088] The computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations, such as fluid handling, correlation of signal to amplicon quantity, and the like. The software converts these instructions to appropriate language for instructing the operation of fluid direction and transport subsystems, light sources or detectors, or the like, as well as for displaying results to a user, e.g., via a display. The computer optionally receives the data from the sensors/detectors included within the system, and interprets the data, either providing it in a user understood format, or to initiate further system instructions, in accordance with the programming, e.g., such as in monitoring and control of system robotics, microfluidic elements, or the like, hi accordance with the present invention, these parameters are optimized for production and detection of nucleic acid amplicons.

[0089] In one aspect, the invention provides software that correlates a plurality of end point signal measurements from different probes that identify different cDNAs in a single reaction to the quantity of one or more of the cDNAs. This software optionally displays the results of the correlation to a user, e.g., on video display, printout, or the like.

Additional Kit Details [0090] The systems and/or kits of the invention can include system or kit instructions (e.g., embodied in a computer or in a computer readable medium, e.g., as system software) for practicing any of the method steps herein. For example, the system optionally includes system software that directs the system to perform any of the method steps noted above, e.g., in one embodiment, the system converts one or more starting RNA into cDNA by reverse transcription, combines separate cDNAs into a PCR mixture and performs PCR on the mixture. The instructions can direct various temperature regulators and/or fluid handlers to move appropriate materials from sources to reaction sites and/or to control reaction conditions (e.g., temperature) at the reaction sites.

[0091] Kits of the invention can include any system of the invention packaged for sale. In addition, kits of the invention can include any composition, reaction mixture, nucleic acid, or reactant for forming the composition, reaction mixture, nucleic acid. The kit components are packaged in appropriate containers and packaged into a kit for sale. The kit can include instructions for performing the methods of the invention, e.g., using the compositions, reaction mixtures, and/or nucleic acids of the invention.

[0092] As noted, the kits optionally include a container or other receptacle in which compositions, reaction mixtures, and/or nucleic acids system component are stored. System components can also be packaged into such receptacles. The elements of the kits of the present invention are optionally packaged together in a single package or set of related packages. The package optionally includes reagents used in the assays herein, e.g., buffers, reverse transcription reagents, amplification reagents, oligonucleotides, standardization reagents, probes, labels, and/or the like, as well as written instructions for carrying out an assay in accordance with the methods described herein. In the case of prepackaged reagents, the kits optionally include pre-measured or pre-dosed reagents that are ready to incorporate into the methods without measurement, e.g., pre-measured fluid aliquots, or pre- weighed or pre-measured solid reagents mat may be easily reconstituted by the end-user of the kit.

[0093] Generally, the systems and kits described herein are optionally packaged to include reagents for performing the systems preferred function. For example, the kits can include any of the system components described along with assay components, reagents, sample materials, control materials, or the like. Such kits also optionally include appropriate instructions for using the system components and reagents, and in cases where reagents are not predisposed in the system components themselves, with appropriate instructions for introducing the reagents into the system. In this latter case, these kits optionally include special ancillary devices for introducing materials into the systems, e.g., appropriately configured syringes/pumps, or the like (in one preferred embodiment, the system itself comprises a pipettor element introducing material into the components of the system Generally, reagents are provided in a stabilized form, so as to prevent degradation or other loss during prolonged storage, e.g., from leakage. A number of stabilizing processes are widely used for reagents that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microcides/bacteriostats, anticoagulants), the physical stabilization of the material, e.g., through immobilization on a solid support, entrapment in a matrix (i.e., a gel), lyophilization, or the like.

EXAMPLES

[0094] The following examples are offered to illustrate, and not to limit the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLE DETECTION OF DIFFERENTIAL EXPRESSION OF THE SOD2 GENE LN WILD-TYPE VERSUS SOD2 +/- MICE.

[0095] In a first embodiment, differential expression of a SOD2 gene in wild-type versus SOD2 +/- mice (heterozygous offspring from a mating between a wild-type mouse and a SOD2 -/- knockout mouse) is measured. Final detection and quantification is performed using quantitative real-time PCR and fluorogenic Taqman probes.

[0096] Fresh liver samples from sacrificed animals are placed into RNAlater

(Ambion), and RNA is extracted from 100 mg tissue using RNAwiz (Ambion) according to the manufacturer's instructions. Contaminating DNA from total RNA is treated with Turbo DNA-free DNase (Ambion) according to the manufacturer's instructions.

[0097] Conversion of total RNA to tagged cDNA is carried out in two separate reactions, one containing SOD2 wild-type total RNA, and the second containing SOD2 +/- total RNA. In a first reaction (SOD2 wild-type total RNA), reverse transcription utilizes gene-specific reverse-transcription (RT) primer #1 :

5 ' -CGTGAGCTGTCACATCCTCGCAGTATGTCAATGTCATCCTCGGTGGCGTTGAGATTGTT-3 '

In a second reaction (SOD2 +/- total RNA), reverse transcription utilizes gene-specific RT primer #2:

5 ' -CGTGAGCTGTCACATCCTCGCAGTATATCGAAGTCTTCCTCGGTGGCGTTGAGATTGTT-3 '

[0098] Reactions are performed using 100 ng of DNA-free total RNA isolated as above and MessageSensor RT reverse transcriptase (Ambion) according to the manufacturer's instructions. Specifically, 20- μl reactions contain 5 μM RT primer #1 or primer #2, and other components according to the manufacturer's instructions.

[0099] The design of the reverse-transcription primers includes three different motifs. The 3 '-most motif is the gene-specific primer, which is complementary to the sequence of mouse SOD2 mRNA and is identical for the two RT primers:

5 ' -CTCGGTGGCGTTGAGATTGTT-3 '

The 5' proximal motif to the gene-specific motif is the tag, which is different for the two RT primers:

Primer #1 : 5 ' -AGTATGTCAATGTCATC-3 ' Primer #2 : 5 ' -AGTATATCGAAGTCTTC-3 '

The 5 '-most motif is a universal motif, which is used in subsequent PCR reactions, and which is identical for the two RT primers:

5'-CGTGAGCTGTCACATCCTCGC-3'

[0100] Following reverse-transcription, the two RT reactions are combined and used as template in a competitive PCR reaction, which is run as a real-time reaction on an ABI 7900HT instrument. This reaction is carried out using the following primer sequences:

Forward Primer:

5'-CAGACCTGCCTTACGACTATGG'3' Reverse Primer:

5'-CGTGAGCTGTCACATCCTCGC-3'

The forward primer is a gene-specific primer complementary to the mouse SOD2 cDNA generated during the RT steps above. The reverse primer is identical to the 5' motif of the two RT primers. The reactions also contain the following two dual-labeled probe sequences:

Probe #1 : 5 ' FAM-GATGACATTGACATACT-NFQ3 ' Probe #2 : 5 ' VIC-GAAGACTTCGAT TACT-NFQ3 '

Where FAM and VIC are the fluorescent dyes and NFQ is a non- fluorescent "Black Hole" quencher (Biosearch). Probe #1 and #2 are complementary to the tag sequences of RT primer #1 and primer #2, respectively, and do not cross-hybridize to each other's sequence under the conditions of PCR described. Therefore, each probe faithfully reports the generation of amplicon from its cognate first-strand cDNA template, and the ratio of FAM versus VIC fluorescence reports the relative amplification of the two differentially-tagged sequences.

[0101] Conditions for PCR are as follows: 20-μl reactions contain IX Universal

Master Mix (Applied Biosystems), 400 nM forward and reverse PCR primers, and 100 nM of probe #1 and probe #2. Cycling is carried out with 15 min x 95 °C, followed by 45 cycles of 15 sec x 95 °C and 60 sec x 60 °C. Fluorescence is captured at each annealing cycle. Fluorescence is normalized relative to the passive reference dye ROX, included in the Universal Master Mix (ABI).

[0102] Finally, comparative analysis is computed by using the end-point fluorescence after the appropriate number of cycles (that point at which amplification has reached a plateau and is no longer increasing). The ratio of F AM/VIC fluorescence is a measure of relative expression of SOD2 +/+ to SOD2 +/- expression. Quantification is made more quantitative through the following additional steps.

[0103] The cycling of the CPR reactions need not utilize a real-time instrument, which is used here merely for convenience. Nor does the analysis require, necessarily, that PCR amplification proceed to a plateau phase. A real-time instrument does provide confirmation that the amplification has achieved a steady state of fluorescence, however, which is likely to minimize variation due to low-abundance fluorescent signal. [0104] First, because the intensity of the two probe reporters is not expected to be equivalent, the relative response of the FAM and VIC probes and RT primers is computed by carrying out the above analysis using the same sample versus both RT primers, thereby permitting a "response factor" for correction of fluorescence difference due solely to the different sequences of the two RT primers, potentially different efficiencies of reverse transcription from these different RT primers, differences in fluorescent output of the probes, and any other incidental differences between the two RT primeπprobe pairs.

[0105] Second, the entire process is also carried out in an inverted orientation, where the SOD2 wild-type sample is paired with RT primer #2 and the SOD2 +/- sample is paired with the RT primer #1. This serves as a control to demonstrate that differences in the relative fluorescence of FAM/VIC are unrelated to the pairing of primer/probe combination with sample and are solely due to differences in transcript abundance of the two samples.

[0106] Third, it will be obvious to those skilled in the art that the above results can be normalized to one of more housekeeping genes, such as beta actin, to correct for differences in the loading of total RNA into the RT reactions themselves. The quantification of transcript abundance for the housekeeping genes can also be performed using the invention disclosed herein.

[0107] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method of quantifying an amplified nucleic acid, the method comprising: (a), reverse transcribing a first nucleic acid in a first reverse transcription reaction comprising a first reverse transcription oligonucleotide comprising a first identification sequence, thereby producing a first cDNA comprising the first identification sequence; (b). reverse transcribing a second nucleic acid in a second reverse transcription reaction comprising a second reverse transcription oligonucleotide comprising a second identification sequence, thereby producing a second cDNA comprising the second identification sequence; (c). combining the first and second cDNAs in a reaction mixture; (d.) amplifying the resulting combined first and second cDNAs in the reaction mixture, thereby producing first and second amplified nucleic acids; and, (e.) detecting the first and second identification sequences in the first and second amplified nucleic acids, wherein the relative quantity of the first and second identification sequences in the amplified mixture provides an indication of the quantity of the first or second nucleic acid.

2. The method of claim 1, wherein the first or second nucleic acid is a messenger RNA, a pathogenic RNA, or a viral RNA, wherein the first nucleic acid is a quantified control nucleic acid and wherein abundance of the second nucleic acid in a sample is a diagnostic marker for one or more gene copy number, infection, disease or condition.

3. The method of claim 1, wherein amplifying the first and second cDNAs comprises performing a PCR reaction with a polymerase, the first and second cDNAs, a first amplification primer complementary to a portion of the first and second reverse transcription oligonucleotides, a second amplification primer complementary to the first cDNA and a third amplification primer complementary to the second cDNA, wherein the third amplification primer is the same or different than the second amplification primer.

4. The method of claim 1, wherein detection of the first and second identification sequences comprises detecting the first or second amplification sequences, or one or more complementary sequences thereof.

5. The method of claim 1, wherein detection of the first and second identification sequences comprises detecting a first probe that specifically hybridizes to the first identification sequence or a complement thereof, and detecting a second probe that specifically hybridizes to the second identification sequence or a complement thereof.

6. The method of claim 5, comprising normalizing a signal detected from the first or second probe to account for one or more difference in activity between the first or second probe, the difference being selected from the group consisting of: (i.) a difference in probe labels that label the first and second probes, (ii.) a difference in amplification efficiency caused by the presence of the first or second identification sequences or complementary sequences thereof in the first or second cDNAs, and, (iii.) a difference in reverse transcription efficiency caused by the presence of the first or second identification sequences in the first or second reverse transcription oligonucleotides.

7. The method of claim 1, the detection comprising end point quantification of one or more probe signals from one or more probes that bind to the first or second identification sequences or complementary sequences thereof.

8. The method of claim 1, the detection comprising end point or real time quantification of one or more probe signals from one or more probes that bind to the first or second identification sequences or complementary sequences thereof, wherein the detecting comprises one or more of: detecting an optical label on the one or more probes, detecting a fluorescent label on the one or more probes, and detecting a luminescent label on the one or more probes.

9. The method of claim 1, further comprising: (f). reverse transcribing the first nucleic acid in a third reverse transcription reaction comprising a third reverse transcription oligonucleotide comprising the second identification sequence, thereby producing a third cDNA comprising the first identification sequence; (g). reverse transcribing a fourth nucleic acid in a fourth reverse transcription reaction comprising a fourth reverse transcription oligonucleotide comprising the first identification sequence, thereby producing a fourth cDNA comprising the first identification sequence; and, (h). comparing an amount of first, second, third, and/or fourth cDNAs, thereby monitoring or controlling for an effect of the first or second identification sequence on the reverse transcription.

10. The method of claim 9, further comprising: (i). combining the third and fourth cDNAs in a reaction mixture; (j). amplifying the resulting combined third and fourth cDNAs in the reaction mixture, thereby producing third and fourth amplified nucleic acids; and, (k). detecting an amount of the first, second, third and first amplified nucleic acids, thereby monitoring or controlling for an effect of the first and second identification sequences on amplification efficiency.

11. A composition, comprising: a first cDNA comprising: a first target sequence complementary to a first RNA sequence of a first RNA isolated from a biological sample, a first amplification primer binding site, and a first identification sequence between the first amplification primer binding site and the first target sequence; wherein the first identification sequence comprises one or more bases that are non-complementary to the first RNA; and, a second cDNA that differs in sequence as compared to the first cDNA, the second cDNA comprising the first amplification primer binding site and a second identification sequence proximal to the first amplification primer binding site, wherein the first and second identification sequences are distinguishable.

12. The composition of claim 11, wherein the first identification sequence is complementary to a molecular beacon or an endonuclease probe.

13. The composition of claim 11, wherein the first RNA sequence is one or more of: an mRNA, a pathogenic RNA sequence, or a viral RNA sequence.

14. The composition of claim 11, wherein the second cDNA comprises a control sequence complementary to a second RNA sequence or an RNA isolated from a biological sample.

15. The composition of claim 14, wherein the control sequence is the same as the first target sequence.

16. The composition of claim 14, wherein the control sequence is different than the first target sequence.

17. The composition of claim 14, wherein the second identification sequence is between the control DNA sequence and the first amplification primer binding site.

18. The composition of claim 11, further comprising one or more of: a polymerase, an amplification primer complementary to the first introduced amplification primer binding site, a dNTP, a first detection probe that specifically hybridizes to the first identification sequence and a second detection probe that hybridizes to the second identification sequence.

19. A kit comprising the composition of claim 14.

20. A reverse transcription primer comprising: a first 3' oligonucleotide subsequence that is complementary to a first subsequence of an RNA isolated from a biological sample; a middle oligonucleotide subsequence complementary to a selected probe sequence, which probe sequence is different than a proximal sequence of the RNA located adjacent to the first subsequence; and, a 5' oligonucleotide sequence that is complementary to the RNA.

21. A reverse transcription reaction comprising the reverse transcription primer of claim 20.

22. A kit comprising the reverse transcription primer of claim 20.

23. A system comprising a pair of reverse transcription reaction mixtures, wherein a first reaction mixture comprises: a first RNA; and, a first transcriptase-extendible oligonucleotide comprising a first transcriptase- extendible subsequence complementary to the first RNA, a first identification sequence, and a first amplification primer binding site, wherein the first identification sequence comprises one or more nucleotides that are non-complementary to the first RNA, and wherein the first identification sequence is between the amplification primer binding site and the extendible subsequence; and, a second reaction mixture comprising a second RNA that is different than the first RNA and a second transcriptase-extendible oligonucleotide partially complementary to the second RNA, the second transcriptase-extendible oligonucleotide comprising a second extendible subsequence complementary to the second RNA, a second identification sequence that is different from the first identification sequence, wherein the second identification sequence comprises one or more nucleotides that are non-complementary to the first or second RNA, and wherein the second identification sequence is between the first amplification primer binding site and the second extendible subsequence.

24. The system of claim 23, wherein the first and second identification sequences are, or are complementary to, a molecular beacon or an endonuclease probe sequence.