WO2013022807A1 - Methods and compositions for detection of nucleic acids using 5'-nuclease cleavage and amplification - Google Patents

Abstract

In some embodiments, methods and compositions are provided to improve the efficiency of 5'nuclease cleavage during PCR by incorporating a short, typically single base, overlap of sequence between the 3' end of a digestion-driving primer and the 5' portion of a probe. A similar overlap can also be used when the primer has an additional 5' portion and spacer between its 5' and 3' ends, such that it can form a unimolecular circular configuration when annealed to the amplicon. In some embodiments, an amplicon forms an intramolecular hairpin structure so that 5' end of a stem overlaps with the 3' end of a digestion-driving primer, when the latter anneals to the amplicon. The amplification reactions can be conducted in solution or with surface-bound primer and/or probes. In some embodiments, methods and compositions are provided to increase the specificity of mutation (allele) specific PCR using blocking oligos cleavable by 5'nuclease reaction.

Description

METHODS AND COMPOSITIONS FOR DETECTION OF NUCLEIC ACIDS USING 5'-NUCLEASE CLEAVAGE AND AMPLIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a non-provisional utility Patent Application claiming priority to and benefit of the following prior provisional U.S. Patent Applications: Ser. No. 61/515,538 filed August 5, 2011, and Ser. No. 61/560,533 filed November 16, 2011, each of which is incorporated herein by reference in its entirety for all purposes.

Background

Various methods are used to detect and quantify DNA in a sample using polymerase chain reaction (PCR). In conventional 5' nuclease assays (marketed as TaqMan™ assays), a probe molecule that includes a fluorescent label and a quencher hybridizes to a PCR amplification product, and is digested by the 5' exonuclease activity of a polymerase. The 5' exonuclease activity releases the fluorescent label from the probe, thereby separating the fluorescent label from the quencher and permitting detection of the fluorescent signal.

U.S. Patent 5,210,015 to Gelfand et al. discloses 5 ' nuclease (TaqMan) assays. It teaches in claim 1 that the 3' end of the probe is annealed upstream of the 5 '-end of the probe: "...a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3' end of the first oligonucleotide is upstream of the 5' end of the labeled oligonucleotide".

Similarly, U.S. Patent 5,487,972 to Gelfand et al. describes the 3' end of the primer being "adjacent" to the 5' end of the probe and "labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the fist oligonucleotide" (primer).

U.S. Patent 5,804,375 to Gelfand et al. describes a "reaction mixture" (composition) that includes two primers and a probe, the probe being "between" the two primers. It also defines the term "adjacent" as follows: "The term 'adjacent' as used herein refers to the positioning of the primer with respect to the probe on its complementary strand of the template nucleic acid. The primer and probe may be separated by 1 to about 20 nucleotides, more preferably, about 1 to 10 nucleotides, or may directly abut one another, as may be desirable for detection with a polymerization-independent process. Alternatively, for use in the polymerization-dependent process, as when the present method is used in the PCR amplification and detection methods as taught herein, the 'adjacency' may be anywhere within the sequence to be amplified, anywhere downstream of a primer such that primer extension will position the polymerase so that cleavage of the probe occurs."

U.S. Patent 5,538,848 to Livak et al. describes 5' nuclease assays requiring an "oligonucleotide probe capable of hybridizing to said target nucleic acid, 3' relative to said primer".

All of the Patents listed above teach that the extension of the digestion-driving primer occurs prior to 5 '-nuclease cleavage. E.g., 5 'nuclease cleavage during PCR in U.S. Patent 5,538,848 is described as follows: "The latter approach, illustrated in Fig. 1, involves the use of an oligonucleotide probe that specifically anneals to a region of the target polynucleotide 'downstream,' i.e. in the direction of extension, of primer binding sites." "During strand extension by a DNA polymerase, the probe anneals to the template where it is digested by the 5'->3' exonuclease activity of the polymerase." "...extending a primer annealed to the target polynucleotide with a nucleic acid polymerase having 5'->3' exonuclease activity such that the oligonucleotide probe is degraded by the 5'->3' exonuclease activity of the nucleic acid polymerase as it extends the primer." Fig. 1 in U.S. Patent 5,538,848 explicitly shows that polymerization/extension and strand-displacement occur before the 5 'nuclease cleavage.

Primer3 is the most widely used software to design PCR primers and probes (Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist

programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols:

Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386). The software always picks the "internal oligo" (i.e., "probe") to be located between and non-overlapping the two amplification primers. Similarly, PrimerExpress software from Life Technologies (www3.appliedbiosystems .com/ cms/ groups/mcb_marketing/ documents/ generaldocuments/ c ms_042505.pdf) recommends to "design primers as close as possible to the probe without overlapping the probe."

Several companies, including Life Technologies, IDT, and Roche/Exiqon UPL offer pre-designed TaqMan (5 'nuclease) assays. In fact, Life technologies offers more than a million pre-designed gene expression assays, several million SNP and CNV assays, and assays for nearly all miR As in human and several other species. As far as one can deduce from information provided by these vendors, no assays have overlaps between the probe and extendable primer. For example, Fig. 1 in

www. appliedbiosystems . com/ cms/ groups/mcb_marketing/documents/ generaldocuments/ cm s_068884.pdf shows digestion-driving primer positioned away from the probe. PrimeTime assays from IDT (www.idtdna.com catalog/primetime/primetime.aspx) explicitly describe 5'nuclease assays having four steps: 1. Annealing; 2. Polymerase extensions; 3. 5'nuclease and 4. Detection of the signal.

In summary, the prior art mentioned above explicitly teaches to avoid overlap between the probe sequence and the extendable primer sequence in PCR. Intuitively this makes sense, since any overlap would create a competition between the primer and the probe: if the 3 ' end of the primer is annealed, the 5 ' end of the probe must be off the template or vice versa. Second, the prior art teaches that polymerase extension is the first step and that it precedes 5'nuclease cleavage.

Increased specificity of allele-specific PCR by using blocking oligos was previously described, e.g., by Zhou et al. (BioTechniques, 2011, 50:311-318) and Tan et al. (J Biomol Tech, 2010, 21(3 Suppl):S30). In both cases, the blocking oligo matches the wild type allele and competes with allele-specific primer to bind to the template: either one, but not both can be bound to the template at the same time.

Nuclease cleavage by Taq polymerase was reported to be more efficient if there is a single base overlap between the 3' end of the primer and the last 5' matching base that is 3' from the 5 '-flap, in a study in which the polymerase did not perform the extension

(Lyamichev et al. PNAS, 1999, 96:6143-6148). The overlap between the probe and an "invading oligo" is used in the invader assays, (e.g., as described in U.S. Patent 5,846,717 to Firmin et al), but the invading oligo merely participates in the formation of a cleavage structure, and does not serve as an extendable primer.

Our Published Application Nos. US 2011/0212846 and WO2011/100057 describe methods and compositions to incorporate universal tags, and then to detect these tags at both ends of the amplicon. The methods described herein can be used for both target- specific and universal detection of encoded targets.

There is a need for improved methods and compositions for detecting and quantifying polymerase-dependent DNA amplification products. Summary

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Rather, the sole purpose of this summary is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented hereinafter.

Described herein are methods and compositions related to 5 '-nuclease during PCR that (a) increase signal generation in qPCR or (b) are using to improve mutation-specific primer specificity using a blocking oligo.

In some embodiments, we provide a 5'nuclease-based PCR method that detects the presence or measures the amount of a target or encoded target nucleic acid.

In some embodiments, the method includes forming an amplicon; annealing a probe comprising a 5 ' portion, and a probe, comprising a 5 ' end region, and a digestion-driving primer, comprising a 3 ' end, to the amplicon; wherein the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the probe by at least one nucleotide when both are annealed to the amplicon, wherein a 5 'nuclease reaction can precede the primer extension of said digestion-driving primer. In some embodiments, the overlap is one nucleotide. In some embodiments, the digestion-driving primer and the probe comprise two parts of a single molecule, wherein the single molecule forms a circular structure when the two parts anneal to the amplicon. In some embodiments, the method includes providing a second primer with a 5 '-probe portion; extending the second primer to form the amplicon, wherein said amplicon comprises a region complementary to the 5' - probe portion; said amplicon forming an intramolecular hairpin stem structure during PCR by annealing of the 5 '-probe portion and said region complementary to the 5 '-probe portion; and, annealing the digestion-driving primer to the amplicon, so that the 3 ' end of the digestion-driving primer overlaps the 5 '-portion of the stem by at least one nucleotide.

In some embodiments, we provide a PCR reaction mixture comprising (i) an enzyme having 5 'nuclease activity; (ii) a digestion-driving primer comprising a 3' end, and (iii) a probe comprising a 5 ' region, wherein the 3 ' end of the digestion-driving primer and the 5 ' region of the probe overlap by at least one nucleotide when both the primer and the probe are annealed to an amplicon formed in said mixture. In some embodiments, the digestion- driving primer and the probe comprise two parts of a single molecule, wherein the single molecule is capable of forming a circular structure when the two parts anneal to the amplicon. In some embodiments, the reaction mixture includes a second primer comprising a 5 '-probe portion, said second primer capable of generating an amplicon having a region complementary to the 5' probe portion; wherein said amplicon is capable of forming an intramolecular hairpin stem structure by annealing of the 5 '-probe portion and the region complementary to the 5 '-probe portion, wherein the digestion-driving primer can anneal to said amplicon so that the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the stem.

In some embodiments, the 5 ' region of the probe when annealed to the amplicon is more thermodynamically stable as compared to the 3 ' region of the digestion-driving primer when it is annealed to the amplicon, so that the structure with 5' probe portion being annealed to the amplicon, favored by 5 'nuclease, is predominant.

In some embodiments, modified nucleotides are used to alter the thermodynamic stability of the 3' region of the digestion driving primer and 5' region of the probe when the primer and probe are annealed to the amplicon.

In some embodiments, the 3 ' end of the primer has a mismatch (non- Watson-Crick pair) or blocked non-extendable 3 ' end when annealed to the amplicon.

In some embodiments, the (a) probe, (b) primer-probe after extension or (c) digestion-driving primer comprise a label and label quencher that are capable of being separated from each other by said 5 'nuclease reaction and generating a detectable signal that allows detection of the presence and/or the amount of target nucleic acid.

In some embodiments, the 5 ' end of the probe comprises a flap sequence tag comprising a label quencher or a label configured to generate a fluorescent or

electrochemical signal when the label quencher or the label are cleaved off together with the flap.

In some embodiments, there is provided a software tool comprising instructions to design primers and probes for methods as described above or for preparing PCR mixtures as described above, said instructions comprising choosing a probe and a digestion-driving primer that match a target nucleotide sequence, or tags in an encoded target sequence, so that there is an overlap of at least one nucleotide between the 5 ' end of the probe and the 3 ' end of the digestion-driving primer.

In some embodiments, a kit includes a digestion-driving primer, a probe or second primer-probe and second primer for use in mixtures as described above, wherein all primers and probes required for a specific application are delivered to customers mixed together or separately and primers and probes can be inventoried, pre-designed or designed when customers submit their target sequences.

In some embodiments, we provide methods to increase specificity of mutation- specific PCR, the method comprising providing a template comprising a putative mutated allele; annealing to said template a blocking oligo having a 5 ' base which matches the mutated allele; and annealing to said template a mutation-specific primer having a 3' end which matches said mutated allele; wherein a 5 'nuclease reaction cleaves the 5' end of the blocking oligo when the mutation-specific primer and the oligo are annealed to the mutated template, but wherein the blocking oligo is cleaved very inefficiently if the template is wild type, thereby further slowing mutation-specific primer extension on the wild-type template.

In some embodiments, we provide a PCR reaction mixture used to detect SNPs or mutations comprising (i) a polymerase with 5 'nuclease activity; (ii) a mutation-specific primer; (iii) a blocking oligo with blocked or otherwise un-extendable 3 ' end; and (iv) template comprising a putative mutated allele, such that the 3 ' end of the primer and at the 5 ' end of the blocking oligo when both are annealed to the template match the mutated allele, and wherein the 5 'nuclease activity is capable of cleaving the 5 ' end of the blocking oligonucleotide so that the primer can extend on a mutant allele, but the 5 'nuclease cleavage is substantially reduced if the template is wild-type, thus providing additional blocking for primer extension. In some embodiments, the mutation-specific primer and blocking oligonucleotide comprises a single molecule that forms a circular structure when annealed to the template.

In some embodiments, in the reaction mixture the digestion-driving primer, probe and/or amplicon (after surface-bound primer extends) and optionally the second primer are attached to a surface during PCR or a bridge amplification and contain at least one label and optionally least one label quencher.

Brief Description of the Drawings

Fig. 1 schematically illustrates a traditional 5' nuclease (TaqMan) cleavage reaction. Fig. 2 schematically illustrates some embodiments of a primer extension reaction. Fig. 3 schematically illustrates an exemplary universal detection format.

Fig. 4 schematically illustrates an exemplary universal circle detection format. Fig. 5 schematically illustrates an exemplary surface-based universal detection format that utilizes bridge PCR and circular primer-probe topology.

Fig. 6 schematically illustrates an exemplary universal circle detection format using primers incorporating flaps.

Fig. 7 shows quantitative PCR data.

Fig. 8 schematically illustrates some embodiments of mutation [allele] -specific

PCR.

Fig. 9 illustrates a flow diagram of some embodiments of a program for primer and probe design.

Description

The present invention is broadly applicable to any application in which one desires to detect or measure the amount of one or more target nucleic acids in a sample of interest.

Provided herein are overlap 5 ' nuclease assay methods and compositions used in PCR in which the sequence at the 3 ' end of a digestion-driving primer overlaps with the sequence at the 5' end of the detection probe. The present invention is broadly applicable to any application in which one desires to detect or measure the amount of one or more target nucleic acids in a sample of interest.

For purposes of simplifying the description of the invention, and not by way of limitation, embodiments in which the digestion-driving primer and probe are separate oligonucleotides will be described initially herein, it being understood that other embodiments are intended to be included within the scope of this invention. Non-limiting examples of such other embodiments are provided below.

Detection formats using separate primer and probe oligonucleotides

The present invention is based in part on the surprising observation by Applicant, as further described below (Example 1 and Fig. 7), that when primers and probes which were intentionally designed to incorporate a single-base overlap between the sequence of the 3 ' end of the primer and the sequence of the 5 ' end of the detection probe were utilized in a quantitative PCR (qPCR) reaction (also referred to as real-time PCR), the amplification signals improved as compared with control reactions using conventional, non-overlapping primers and probes. The current dogma for qPCR detection is that the method always produces relative data: delta C_t values are used to compare samples to each other or to the "standard curve" and quantification is different for each assay. Conventional TaqMan methodology (see U.S. Patent 5,487,972 and 5,804,375) is considered the "gold standard" for qPCR. Three factors cause TaqMan quantification to be relative: (1) kinetics of probe annealing is often slow, so a significant percentage of extension of the digestion-driving primer occurs prior to probe annealing; (2) the polymerase displaces the TaqMan probe, rather than cleaving it; and (3) PCR does not double at cycles prior to C_t. One can estimate that only 5-60% (depending on the assay) of amplicons generated in a TaqMan reaction produce probe cleavage. We believe that intramolecular detection methods described previously, (see, e.g., U.S. Patent 6,326,145 to Whitcombe et al. and our Published Application No. US 2011/0212846) together with the novel methods and compositions described herein will disrupt the current dogma and make qPCR closer to being an absolute: a specific C_t value measure by an instrument indicating a certain number of target molecules present in the sample.

Without intending to be limited by theory, it is noted that without this overlap, the polymerase has to complete three steps, each potentially lowering the efficiency of nuclease cleavage. DNA polymerases used in PCR are bi-functional molecules having both polymerization (P) and 5 ' nuclease (N) active sites, which are contained in separable domains. Thus, a polymerase may be depicted as having a polymerase active center (shown as a dashed circle in Fig. 1 and 2) and a 5' nuclease active center (shown as a solid circle in Fig. 1 and 2) oriented towards the junction of the 3' end of the primer and 5' end of the probe. Referring to Fig. 1, it is theorized that in the course of extension of the digestion- driving primer, the polymerase (i) has to displace the 5 ' most matching base of the probe and make a base extension to generate a one -base overlap (Fig. 1(a) and 1(b)), (ii) the 3' end of the primer needs to move away from the template so that the 5 ' base of the probe can anneal back to the template (Fig. 1(d)) and (iii) the polymerase has to reorient so that its nuclease active center faces the 5' end of the probe (Fig. 1(d)) in order to perform the cleavage (not shown). Cleavage can only occur if the 5' end nucleotide is annealed to the template. In some circumstances, the polymerase does not have sufficient strand

displacement activity, and could stall on step (i) above which would decrease the overall efficiency of PCR. Additionally, instead of performing the last two steps above, the polymerase would have an easier time by merely continuing the extension, with

displacement of the 5 '-end of the probe (Fig. 1(c)), rather than performing cleavage because the polymerase would not have to reorient its active center. The overlap region of the 3 ' end of the primer and the 5 ' end of the probe can form two different structures when annealed to the template. It is expected that the structure in Fig. 1 (b), in which the 3 ' end of the primer is annealed and the 5 ' end of the probe is off of the template, will have a higher affinity toward the polymerase active site of the enzyme. The alternative structure with the 5 '-end of the probe annealed and the 3 ' end of the primer off the template (Fig. 2(a)) will have a higher affinity for the 5 ' nuclease active site of enzyme (Fig. 2(b)), thus facilitating cleavage by avoiding the extension and displacement steps of Figs. 1(a) and 1(b). The extension can start only after the 3 ' end of the primer anneals to the template (Fig. 2(c)).

It is believed that the sequence overlap between the digestion-driving primer and probe, as described herein, favors 5 'nuclease cleavage as a requisite first step in the qPCR, which precedes primer extension, and which will lead to an increase in the efficiency of nuclease cleavage during qPCR. This increase in efficiency would account for the improved qPCR performance as observed herein.

In some embodiments, one or more nucleotides are designed such that at the 5 ' end of the probe is more thermodynamically stable in binding to the template than the nucleotides at the 3' end of the primer (the two are competing to anneal to the same base[s] on the template) in order to promote the structure in Fig. 2(a) and thus promote 5 'nuclease activity over extension.

In some embodiments, a dinucleotide at the 5 ' end of the probe can be designed to be more thermodynamically stable than the dinucleotide at the 3 ' end of the primer. For example, in the case of a single base overlap, the 3 '-most primer dinucleotide WN-3' (W=A or T) would be less thermodynamically stable in binding to template than the 5 ' most probe dinucleotide 5'-NS (S=G or C), where N is the same single overlapping base. In addition to relying on thermodynamic properties of dinucleotides, one can introduce duplex stabilizing bases in the 5' region of the probe where nuclease cleavage is to occur and/or introduce duplex destabilizing bases close to the 3 ' end of the primer. Examples of duplex-stabilizing bases include, but are not limited to, C-5 propynyl-dC (pdC), 5-methyl-dC or AP-dC that can substitute for "C", pdU for "T" and 2-amino-dA for "A". One can weaken primer binding by using 4-Et-dC or inosine. Amidites for all the modified bases mentioned above are available from Glen Research. These modified bases increase the initial cost of primers and probes, but the additional cost may be amortized over a large number of experiments, for example if one is using universal detection methods such as those described in Published Application No. US 2011/0212846. In some embodiments, probes that overlap with primers can use modified bases, for example, LNA (locked nucleic acids) are currently used by UPL (universal probe library) from Exiqon/Roche. In some embodiments, probe-stabilizing moieties may be used, such as, MGB (minor groove binder from Epoch/Life technologies) or BHQplus (BioSearch).

In some embodiments, a modification is introduced at the 3 ' end of the primer which overlaps the 5 ' end of the probe, such that polymerase cannot extend or extends

inefficiently. For example, a mismatch base at the 3 ' end of a primer would cause the 3 ' end of the primer to be less thermodynamically stable than the 5' end of the probe thus promoting the structure shown in Fig. 2(a). The polymerase would be very inefficient in extending such a mismatch, while the 5 'nuclease activity would not be affected by the 3' mismatch (Lyamichev, 1999). After the 5 'nuclease cleavage, the 3 '-exonuclease

(proofreading) activity of polymerase removes the mismatch, allowing primer extension without incorporating the mismatch into the amplicon, so that this process is repeated at every cycle. Alternatively, a base modification can be incorporated at the 3' end of the primer, e.g., pyrophosphate, that prevents primer extension until the blocking moiety is removed.

Probes can include one or more than one quencher. For example, ZEN probes from IDT include both a 3 '-quencher and a second internal ZEN quencher closer to the middle of the probe that decreases the background fluorescence.

According to some embodiments, we provide herein methods for the detection of a target nucleic acid sequence in a sample, said methods comprising:

(a) contacting a sample comprising single-stranded nucleic acids with a first oligonucleotide containing a sequence complementary to a region of the target nucleic acid and with a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid strand to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3' end of the first oligonucleotide overlaps the 5' end of the labeled oligonucleotide by at least one nucleotide;

(b) maintaining the mixture of step (a) with a template-dependent nucleic acid polymerase having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments; and

(c) detecting and/or measuring the release of labeled fragments. In some embodiments, the 3' end of the first oligonucleotide in the annealed duplex of step (a) overlaps the 5' end of the annealed, labeled oligonucleotide by one nucleotide.

Intramolecular detection formats

The nucleotide overlaps described herein may be readily introduced in universal assays such as described in our Published Application No. US 2011/0212846 (herein incorporated by reference in its entirety), as well as traditional target-specific 5 'nuclease (aka TaqMan) assays described above.

In some embodiments, the present invention is directed to methods and

compositions for detecting and/or measuring the amount of a target nucleic acid in a sample using a set of universal PCR primers with sequences that do not hybridize to the target nucleic acid of interest. In some embodiments, the invention provides a method for detecting a target nucleic acid that includes tagging the target nucleic by linking first and second universal segments at the two ends of a molecule in a way that depends on the target nucleic acid, and PCR amplifying the tagged target nucleic acid using universal primers that hybridize to the universal segments. In these embodiments, regions of the PCR amplicons corresponding to the first and second universal segments form intramolecular hairpin stems, where hairpin stem formation is a prerequisite for detection of the nucleic acid, e.g., by removal of a label, label quencher and/or FRET dye from the amplicon. These and other embodiments of the invention are described in detail herein below.

In some embodiments, there are provided methods and compositions for detection of nucleic acids that utilize artificial universal tagging sequences rather than traditional direct detection of DNA/RNA using primers and probes matching the target nucleic acids. Target nucleic acids are only used to specifically connect distinct universal tagging sequences into a single molecule during the first encoding step. The detection is based on the intra-molecular Watson-Crick base-pairing (a hairpin stem) between the two universal tags in the amplicon. The base-pairing between the two universal tagging sequences enables detection of nucleic acids that preserves "three tag" detection specificity: spurious amplifications and primer dimers have a low probability of forming hairpins. Also provided is universal surface solid-phase detection for encoded nucleic acid targets.

A variety of universal detection formats are provided herein. Some embodiments are schematically illustrated in Fig. 3. As shown, step (a) includes priming on both strands of a first tagged nucleic acid using first universal primer 300 and second universal primer 302. A 3' portion (Ai) of the first universal primer anneals to first universal segment 304, while a 3' portion (Ci) of the second universal primer anneals to portion C'i of second universal segment 306. The first universal primer includes detectable label Dl at or near the 5' end of the first universal primer, 5' portion (Bi) and a label quencher or FRET dye (Q). Optionally positioned proximal to the label quencher or FRET dye is a polymerase blocking unit, e.g., HEG (hexethylene glycol), or any other blocker known in the field in case the presence of the quencher or FRET dye is not sufficient to stop the polymerase extension. For example, primers with a HEG or Sp-18 polymerase blocking unit are commercially available, e.g., from BioSearch Technologies. Some quenchers can also serve as polymerase blockers, e.g., ZEN from IDT. For the sake of simplifying the figures herein, polymerase blocking units are not shown. However, it will be understood that a polymerase blocking unit is optionally positioned in proximity to quencher "Q" in the middle portion of the universal primers. A polymerase blocker may not be required if the 5 '-tail that folds into a stem has one or more bases at the 5' end that are not complementary to the middle universal tag sequence, so that the hairpin formed by the opposite strand of DNA (with the 3 '-end at the end of the stem) is not extendable during PCR. One can also design a small hairpin into the 5' portion of the primer 300, so that the dye and the quencher are brought closer together, similar to

"Sunrise" primers and probes, to improve quenching and decrease background fluorescence. For example, see U.S. Pat. Nos. 5,866,336 and 6,270,967.

PCR amplification results in double stranded product 308. In this example, a polymerase blocking unit positioned proximal to the label quencher or FRET dye prevents a polymerase from copying the 5' portion (Bi) of the first universal primer, such that the bottom strand of product 308 cannot form a hairpin when it becomes single-stranded.

Formation of such a hairpin would result in the 3' end of the stem annealing to the amplicon such that polymerase extension of this 3' end would terminate the PCR reaction.

Hairpin formation is shown at step (c) of Fig. 3. Product 308 is melted (e.g., by raising the temperature to approximately 95°C.) to separate the upper strand from the lower strand, and when the temperature is subsequently decreased, the upper strand of product 308 forms a hairpin having a stem between 5' portion (Bi) of the first universal primer and portion B'i at the opposite end of the strand. Then, at step (c), the second universal primer Ci anneals to a complementary portion of the upper strand. The 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides. In some embodiments, the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide. Intra-molecular hairpin formation occurs rapidly and is driven by thermodynamics: the free energy is determined by stem length, GC-content and loop length. It is important that the melting temperature (Tm) of the hairpin be significantly higher (e.g., approximately 10°C. or higher) than the Tm of the second universal primer 302. This way, when the temperature is decreased, nearly 100% of the molecules will form the hairpin before the second universal primer anneals. After annealing the second universal primer at step (d), 5' nuclease activity cleaves the detectable label Dl from the 5' end of the amplicon, thereby increasing the distance between the label and the quencher or FRET dye and permitting detection of the label. A wide variety fluorescent dyes are known in the art and commercially available, e.g., FAM, TET, JOE, VIC, HEX, CY3, TAMRA, TexasRed, CY5, ROX and many other dyes and quenchers can be used, e.g., Dabsyl, MGB-NFQ, BHQ-[0123], Iowa Black, ZEN quencher from IDT, and others.

In some embodiments, we provide a detection scheme similar to that shown in Fig. 3, but where the positions of the detectable label and quencher or FRET dye on the first universal primer are switched. In some embodiments all primers in Fig. 3 are target- specific, and the formation of an intramolecular hairpin stem is similar to that described in U.S. Pat. No. 6,326,145.

Some embodiments of a detection format involving the formation of a universal circle are schematically illustrated in Fig. 4. The tagged target nucleic acid is amplified using universal primer 400 (including Dl-Bi-Q-spacer-x-Ci-3') and second universal primer 402 (including Ai), where Dl is a detectable label, Q is a quencher and "x" is a polymerase blocking unit. A 3' portion (Ci) of the first universal primer anneals to first universal segment 404, while a 3' portion (Ai) of the second universal primer anneals to second universal segment 406. As shown, primer 400 is designed such that the 3' end of the primer overlaps with its own 5' tail (forming a circular structure). In this example, the first universal primer Ci at step (b) generates a signal by 5' exonuclease cleavage of Dl and separation of Dl from the quencher, and then extends.

Some embodiments of a surface-based universal detection format provided by the present invention are schematically illustrated in Fig. 5. In these embodiments, universal detection is accomplished via "bridge PCR" or bridge amplification, where the universal primers are attached to the surface of a bead. As shown, first universal primer 500 and second universal primer 502 are attached to the surface of bead 504. The first universal primer includes Q-Bi-Dl-spacer-[surface]-spacer-Ci-3' and the second universal primer includes Ai, where Q is a label quencher and Dl is a detectable label, e.g., a fluorescent dye. First tagged target nucleic acid (including Ai-B'i-C'i) anneals to both the Bi and Ci portions of the first universal primer, bringing the 3 '-end of Ci close to the 5 '-quencher of Bi. The 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides. In some embodiments, the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide. The Ci at step (c) cleaves the quencher and generates fluorescent signal Dl . At step (d), the extended amplicon forms a bridge by looping back onto Ai on the surface, and Ai extends to form a complementary strand, thus continuing amplification on the surface. The first universal primer on the surface is shown attached in the middle. An alternative configuration in accordance with the present invention would be to have a spacer-Ci-3' primer attached to the bead at the 5' end and Q-Bi-Dl-spacer-5' attached at the 5' end. The ends of the two attached primers would come into proximity with each other due to their attachment to the same bead surface. In this example, the two parts of the first universal primer are formally two separate oligonucleotides, but being bound to the same bead surface they are effectively linked into a single molecule where the surface provides a link. In other embodiments, Ai primer can be in solution (not surface bound), so that only a single strand with the label is bound to the surface. The same Ai solution primer can be used by surfaces with different B- and Ci tags.

Multiple beads with different intrinsic bead properties can be used for multiplex detection in Fig. 5. For example, bead color (Luminex microspheres), holographic images (Illumina VERACODE™) or barcodes (Applied BIOCODE™ beads, Affymetrix liquid arrays) can be pooled together for the detection step. One can decode both the universal tags "i" on each bead that encode each target based on the intrinsic bead properties and the surface signal generated by the label on the first universal primer that measures the amount of the tagged target nucleic acid in the sample.

Surface detection for multiplex targets requires attaching universal primers to beads or surfaces (Fig. 8(a)). Bead encoding can be performed by the bead vendor: each type of bead with different colors or barcodes is combined with specific tags Bi, Ci and optionally Ai. All encoded beads are pooled together for multiplex detection and the universal pool can be used to detect or measure any nucleic acid target. Users can mix their samples containing target nucleic acids, encoding primers, master mix and encoded beads and PCR cycle the mixture. First, encoded target molecules will be generated and then these molecules anneal to the surface bound universal primers and generate a signal on the surface (see, e.g., Fig. 5). In an alternative two-step detection method, targets are first tagged by linking universal segments together. The tagged/encoded sample is optionally diluted and added to the pooled decoding beads for detection. After hybridization to the beads, bridge amplification or PCR generates signal on the bead surface. For the readout, the beads can be laid on a surface (e.g., VERACODE™), streamed past a detector (e.g., Luminex microspheres) or directly imaged in a well (e.g., Applied BIOCODE™). The barcode on a bead or color of a microsphere determines the target and the fluorescent signal on the surface measures the amount of this nucleic acid target in the sample. Thus, as provided by the present invention, multiplex surface detection can measure the amounts of multiple targets in a well using a set of universal beads or microspheres.

It will be appreciated that the universal detection formats described above are exemplary approaches. Additional universal detection formats are possible and within the scope of the present invention. It will also be appreciated that any of the exemplary universal detection formats described above can be combined with any of the universal segment linking strategies and universal detection applications described herein. Further, it will be understood that the universal detection formats of the present invention can occur in multiplex, where 2 or more, e.g., 4 or more, 6 or more, 8 or more, 10 or more, hundreds or more, or even thousands or more different target nucleic acids of interest can be detected simultaneously in wells (e.g., nano-wells), on beads, on an array, using integrated fluidics chips (IFC), and the like.

Some embodiments of universal detection formats are schematically illustrated in Fig. 6. As shown, step (a) includes priming on both strands of a first tagged nucleic acid using first universal primer 66 and second universal primer 68. A 3' portion (Ai) of the first universal primer anneals to portion Ai of a first universal segment 62, while a 3' portion (Ci) of the second universal primer 68 anneals to portion C'i of second universal segment 64. The first universal primer includes flap Fli and detectable label La at or near the 5' end of the flap, 5' portion (Bi) and a label quencher or FRET dye (Q). The second universal primer includes flap F2i and detectable label La at or near the 5' end of the flap, 5' portion (Ei) and a label quencher or FRET dye (Q).

Hairpin formation is shown after step (b) of Fig. 6. PCR product is melted (e.g., by raising the temperature to approximately 95°C.) to separate the strands, and when the temperature is subsequently decreased, one strand of product forms a hairpin having a stem between 5' portion (Bi) of the first universal primer and portion B'i at the opposite end of the strand and the other strand having a stem between 5' portion (Ei) of the first universal primer and portion E'i at the opposite end of the strand. The universal primer Ci anneals to a complementary portion of one strand. The 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one or more nucleotides. In some embodiments, the 3' terminus of primer Ci overlaps the 5' terminus of portion Bi by one nucleotide. Similarly, the 3' terminus of primer Ai overlaps the 5' terminus of portion Ei by one or more nucleotides. 5' nuclease activity cleaves the flaps allowing signal generation.

Mutation or Allele Specific PCR

The ability to detect low levels of sequence variants among mostly wild-type DNA is critical for early cancer detection, prenatal testing, and infectious diseases. In cancer, low- level mutations (<10%) are usually below the detection limits of most genotyping techniques, including standard sequencing.

In some embodiments of the present invention, there are provided improved methods for enrichment and detection of rare alleles which take advantage of the interplay between the 5 ' nuclease activity and the polymerse activity of DNA polymerase in order to increase the specificity of mutation (allele)-specific PCR. The present methods provide a novel means to prevent mutation specific primers from occasionally extending a 3 ' mismatched nucleotide on the "wild-type" alleles, and thereby substantially avoid false positive readings. In some embodiments, a blocking oligonucleotide (probe) is used with its 5 ' nucleotide matching the mutation in the same reaction with an allele-specific blocking oligonucleotide.

In Fig. 8, an allele-specific primer has an arrow at the 3 '-end. The blocking oligonucleotide or tag is depicted as a double-line. Fig. 8(a) shows blocking on a wild-type amplicon, Figs. 8(b) and 8(c) show 5 'nuclease cleavage followed by primer extension on a mutated amplicon. As shown in Fig. 8(a), when both the primer and the oligonucleotide are annealed to the wild-type allele, the 5 'nuclease reaction is inhibited because of the 5' nucleotide mismatch in the blocking oligonucleotide. Without wishing to be bound by theory, it is believed that there is a redundant inhibition in the polymerase extension due to both the 3 ' mismatch in the primer, as in conventional allele-specific PCR but, in addition, because extension requires strand-displacement of the 5' region of the oligonucleotide. The 5' nucleotide of the blocking oligonucleotide remains un-cleaved, and provides additional blocking in the presence of a wild-type allele. By contrast, when there is no mismatch (Fig. 8(b)), the 5 'nuclease reaction will readily cleave off the 5' nucleotide on DNA templates with mutations, and polymerase will then extend the primer, displacing the rest of the blocking oligonucleotide off of the template (Fig. 8(c)). In preferred embodiments, the blocking oligonucleotide has a non-extendable 3' end; either a blocking moiety, e.g., 3 'phosphate, amine, a quencher, C3, etc. or mismatch bases can be used (shown as "X" in Fig. 8(a)-(c)). Typically, the blocking oligo has a higher Tm that the mutation-specific primer, so that it anneals before the primer. The blocking methods as disclosed herein differ from other blocking methods (e.g., Zhou et al., 2011 and Tan et al., 2010) in at least two ways: (a) both allele-specific primer and blocking oligonucleotide are bound to the template concurrently rather than competing with each other, so that only one of the two can bind at any given time; (b) the blocking oligonucleotide matches the mutation, rather than the wild- type as in the other methods.

Some embodiments utilize a unimolecular format, in which the function of the blocking oligonucleotide is performed by the 5 '-tail part of the allele-specific primer itself, when it anneals to the template forming a circle, so that the 3 ' and 5 ' ends of the allele- specific primer match the same mutated allele on the template (Figs. 8d, 8e). In preferred embodiments, a blocking moiety, e.g., HEG (AKA SP18), is present in the region of the spacer of circular allele-specific primer ("X" in Fig 8d-f), so that it continues forming a circle, rather than priming, during later cycles of PCR.

Overlap 5 'nuclease assays can be used for all nucleic acid detection applications, including, but not limited to, (1) SNP/indel/MNP genotyping or allelic expression, (2) mRNA (gene expression) including splice junctions and fusions, (3) copy number variation, (4) miRNA or other non-coding RNA, (5) methylation, (6) bacteria, viral or fungal or any species, (7) mutations, including somatic (8) translocations, inversions, insertions, deletions or other large-scale genomic variations, and (9) nucleic acids treated with bisulphate, nucleases or restriction enzymes. In the case of encoding followed by universal detection, one can detect several targets together using the same signal/dye, e.g., any mutation that causes resistance to a given drug, or a set of somatic mutations. Examples are described in our Published Application No. US 2011/0212846 and more examples are provided in U.S. Patent Application No. 61/515,538.

The read-out for qPCR reaction can be real-time: signal measured at many or every cycle or end-point signal detected at the end of qPCR. The latter can also be used for digital counting, so called digitalPCR: counting number of positive (1, 2, 3 etc. copies of [encoded] target DNA) and negative droplets, beads or wells, and applying Poisson statistics to accurately approximate the number of [encoded] DNA target molecules in the sample.

Higher percentage of 5 'nuclease cleavage when using the methods and compositions described herein, and as demonstrated in the below Example and Fig. 7, provides several important advantages compared to traditional 5 'nuclease (TaqMan) assays: (1) increased sensitivity and earlier signal detection (2) higher signal to noise ratio as signal increases, but where the noise stays the same; (3) higher precision in measuring C_t values in qPCR because more data points with exponential growth are used to approximate C_t while the signal is doubling.

The unimolecular interaction (hairpin or circle formation) described in the US Patent 6,326,145 and Published Application No. US 2011/0212846 is kinetically much quicker than probe annealing, i.e., the 5 'end of amplicons or primers will nearly always anneal before the 3' end of the primer. The primer/probe overlap, as described herein, will promote the occurrence of nuclease cleavage prior to primer extension and probe displacement. The combined use of these two improvements can lead to close to 100% 5 'nuclease cleavage: every amplicon generated by PCR will produce a cleaved molecule. PCR doubling during early cycles can usually be achieved by optimizing the PCR master mix. Thus, we believe that the methods and compositions described herein will challenge current qPCR dogma and make nucleic acid detection close to absolute: every assay will have a similar relationship between the number of target molecules in the sample and C_t (or other signal used for detection). We believe that the increased signal (sensitivity) and absolute quantification will provide significant improvements to all qPCR detection application areas, including research, applied markets, and diagnostics.

Primer and Probe Design Software

In some embodiments, a new software tool is provided herein which allows the user to incorporate overlaps between the probe and the associated primer.

In some embodiments, there are provided herein methods for designing a PCR primer and probe for amplifying a target sequence. In some embodiments the methods include applying criteria for designing a candidate primer and a candidate probe for amplifying the target, wherein the criteria include designing the 3 ' end of the primer to overlap the 5 ' end of the probe by at least one nucleotide. The methods can also include receiving and storing a sequence of a target nucleic acid to be amplified by the primer and probe; and storing and/or displaying the sequence of the candidate primer and probe. The methods can include transmitting the sequence of the candidate primer and probe.

Fig. 9 is a flow diagram of some embodiments of a computer implemented process 900 for generating candidate PCR primers and probes for use in the methods disclosed herein. At step 910, a user interface (e.g., form) is generated where a user can specify target sequences, and use default parameters or optionally fields to input parameters such as, minimal/optimal/maximum primer lengths, product size, primer Tm, %GC, target regions, and other parameters. The target sequence is received (i.e., entered by the user) at step 920 and stored. The parameters, including start and stop, are received at step 930. In step 940, primer parameters are optionally received. In step 950, primer and probe overlap parameters are received. These parameters can include, in some embodiments, the number of overlapping nucleotides between the 3 ' end of the primer and the 5 ' end of the detection probe, which can be selected as 1, 2, 3 etc. In step 960, the user clicks a "Pick Primer" key or similar selection key, and the program scores and displays candidate forward primers, candidate reverse primers, and probe sequences. In step 970, the program scores and displays candidate primer and probe sequences.

Currently available software tools used to design 5 'nuclease assays specifically prevent overlaps between primers and probe. Traditional primer/probe design software tools pick multiple forward, reverse primer and probe candidates independently and then find combinations that provide better scores based on multiple criteria: primer/probe-Tm, complementarity between oligos, primer/probe/amplicon lengths, etc. Primer3 software documentation, for example, describes multiple criteria used for primer/probe design. The overlap primer/probes design methods, as disclosed herein, are different: first multiple candidate digestion-driving primer and probe pairs with a desired overlap are picked and scored. Then multiple 2^nd primer candidates are scored by themselves and against primer/probe pairs based on the criteria that are similar to those traditionally used for primer probe design. When using a hairpin amplicon design, the 2^nd primers are spliced with the probe with an optional polymerase blocker in-between. As with conventional primer probe design, the probe can match either of the two DNA strands and thus it can be paired with either the forward or the reverse primer. One can also allow small overlaps between the detection probe and the reverse (non-digestion-driving) primer, provided this overlap is too short to cause reverse primer extension on the probe. The reverse primer primes on the strand different from the probe, so this is only a "computational" and not a real overlap.

Kits

Also provided herein are reagents and kits suitable for carrying out an amplification as described herein. Such kits can include reagents suitable for carrying out qPCR amplification reactions. In some embodiments, the kit comprises forward and reverse amplification primers suitable, as described herein, for amplifying a target polynucleotide of interest and a detection probe. In some embodiments, the 3' end of the forward primer and the 5 ' end of the probe are designed to have an at least one nucleotide overlap when bound to a template as described herein. In some embodiments, the overlap is two nucleotides. In some embodiments, the overlap is one nucleotide.

In some embodiments, a kit includes at least one DNA polymerase, dNTPs, and/or buffer, suitable for carrying out template-dependent primer extension. In some

embodiments, the probe is labeled with a labeling system suitable for monitoring amplification of a target polynucleotide as a function of time. A kit can include written directions for carrying out methods as described herein. A kit can include software for designing primers and probes as disclosed herein and/or instructions for accessing and using website implementing such software. In some embodiments, a kit can include means for detecting amplification products.

Target-specific overlap 5 'nuclease assays using probes, hairpins, or circles can be offered to customers as (a) designed when customer submits targets (AKA "assays-by- design"); (b) pre-designed for a set of popular targets, e.g., RefSeq mRNAs, and (c) inventoried assays, e.g., for miRNA targets. Assays can be provided to customers preloaded in plates, cartridges, or beads / surface-attached. The universal overlap 5' nuclease assays (e.g., as described in Published Application No. US 2011/0212846) would be inventoried and can be offered preloaded in plates, cartridges, and beads or other surfaces.

Overlap 5 'nuclease assays can be used for research use, applied markets, e.g., pathogen detection in food, bio-production reactors, human identification, forensics, environmental testing, epidemiology, etc., veterinary, and human diagnostic use.

Target Nucleic Acid Sources and Molecular Biology Reagents and Techniques

As will be appreciated, target nucleic acids that find use in the invention can be obtained from a wide variety of sources. For example, target nucleic acids can be obtained from biological or laboratory samples including cells, tissues, lysates, and the like.

In certain aspects, the source of target nucleic acids includes cells or tissues from an individual with a disease, e.g., cancer or any other disease of particular interest to the user. A plethora of kits are commercially available for the purification of target nucleic acids from cells or tissues, if desired (see, e.g., EASYPREP™, FLEXIPREP™, both from

Pharmacia Biotech; STRATACLEAN™ from Stratagene; QIAPREP™ from Qiagen). In addition, essentially any target nucleic acid can be custom or standard ordered from any of a variety of commercial sources.

General texts which describe molecular biological techniques for the isolation and manipulation of nucleic acids include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and 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 the current date) ("Ausubel")).

Labeling strategies for labeling nucleic acids and corresponding detection strategies can be found, e.g., in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals Sixth Edition by Molecular Probes, Inc. (Eugene Oreg.); or Haugland (2001) Handbook of Fluorescent Probes and Research Chemicals Eighth Edition by Molecular Probes, Inc. (Eugene Oreg.) (Available on CD ROM).

A number of embodiments of the present invention utilize the principles of polymerase chain reaction (PCR). PCR methods and reagents, as well as optimization of PCR reaction conditions (e.g., annealing temperatures, extension times, buffer components, metal cofactor concentrations, etc.) are well known in the art. Details regarding PCR and its uses are described, e.g., in Van Pelt-Verkuil et al. (2010) Principles and Technical Aspects of PCR Amplification Springer; 1st Edition ISBN-10: 9048175798, ISBN-13: 978- 9048175796; Bustin (Ed) (2009) The PCR Revolution: Basic Technologies and

Applications Cambridge University Press; 1st edition ISBN-10: 052188231 1, ISBN-13: 978-0521882316; PCR Protocols: A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Chen et al. (ed) PCR Cloning Protocols, Second Edition (Methods in Molecular Biology, Volume 192) Humana Press; and in Viljoen et al. (2005) Molecular Diagnostic PCR Handbook Springer, ISBN

1402034032.

Amplification reactions may be carried out with a variety of different DNA polymerases (or mixture of DNA polymerases), but are preferably carried out in the presence of one or more thermostable polymerases. Suitable thermostable polymerases include, but are not limited to, Taq and Tth polymerase (commercially available from Life Technologies). Moreover, like conventional RT-PCR amplification reactions, RT-PCR amplification reactions may be carried out with a variety of different reverse transcriptases (or mixture of reverse transcriptases), although in some embodiments thermostable reverse- transcriptions are preferred. Suitable thermostable reverse transcriptases include, but are not limited to, reverse transcriptases such as AMV reverse transcriptase, MuLV, and Tth reverse transcriptase.

In some embodiments, the polymerase is selected from the group consisting of Taq DNA polymerase, Taql DNA polymerase, Thr DNA polymerase, Tfl DNA polymerase, Tru DNA polymerase, Tli DNA polymerase, Tac DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, rTth DNA polymerase, Pfu DNA polymerase, Pho DNA polymerase, Pwo DNA polymerase, Kod DNA polymerase, Bst DNA polymerase, Sac DNA

polymerase, Sso DNA polymerase, Poc DNA polymerase, Pab DNA polymerase, Mth DNA polymerase, ES4 DNA polymerase, VENT™ DNA polymerase (New England Biolabs), DEEPVENT™ DNA polymerase (New England Biolabs), PFUTurbo™ DNA polymerase (Stratagene), AmpliTaq DNA polymerase (Life Technologies), Tth DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, AmpliTaq Gold DNA polymerase (Life

Technologies), KlenTaq-1 polymerse, E. Coli DNA polymerase I, Klenow fragment of E. Coli DNA polymerase I, SEQUENASE™ 1.0 DNA polymerase (Amersham Biosciences), SEQUENASE 2.0 DNA polymerase, and mixtures thereof.

Temperatures suitable for carrying out the various denaturation, annealing and primer extension reactions with the polymerases and reverse transcriptases are well-known in the art and/or can be determined empirically.

As noted herein, the universal detection steps of the present invention can be performed in real-time, e.g., where one or more detectable signals (if any) corresponding to the presence or amount of one or more target nucleic acids are detected at the conclusion of one or more PCR cycles prior to completion of thermal cycling. Real-time/quantitative PCR techniques are known in the art. Detailed guidance can be found in, e.g., Clementi M. et al (1993) PCR Methods Appl, 2: 191-196; Freeman W. M. et al (1999) Biotechniques, 26: 112- 122, 124-125; Lutfalla G. and Uze G. (2006) Methods Enzymol, 410: 386-400; Diviacco S. et al (1992) Gene, 122: 313-320 Gu Z. et al (2003)/Clin. Microbiol, 41 : 4636-4641. Realtime (e.g., quantitative) PCR detection chemistries are also known and have been reviewed in, e.g. Mackay J., Landt O. (2007) Methods Mol. Biol, 353: 237-262; Didenko V. V.

(2001) BioTechniques, 31, 1106-1121; and Mackay L M. et al (2002) Nucleic Acids Res., 30: 1292-1305, which are incorporated herein by reference in their entireties for all purposes. The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the presently disclosed compositions and methods, and are not intended to limit the scope of what the Applicant regards as the invention nor are they intended to represent that the experiments below are all or the only experiments performed.

Example 1

Effect of Primer/Probe Single Base Overlap on Quantitative PCR Amplification

Fig, 7 compares 5 'nuclease amplification curve signals for (a) probe-based

(TaqMan) 5 'nuclease assays and (b) hairpin forming amplicon formats (as shown in Fig. 3). The FAM signal is on the Y-axis; PCR cycle number on the X-axis. Primer, probe and artificial template sequences are shown in the Sequence Listing below. The six

amplification curves correspond to three dilutions (10³, 10⁴ and 10⁵ copies) and "overlap" ("o"; corresponding to curves 700', 710' and 720', respectively) and "non-overlap" ("n"; corresponding to curves 700, 710 and 720, respectively) assay designs. The two designs were identical, except the "overlap" designs had an additional base at the 3' end of the primer: compare SEQ ID NO: 2 for the probe assays (a) and SEQ ID NO: 7 and SEQ ID NO: 8 for the hairpin. In order to keep the same primer lengths, an extra base was added at the 5' end of the "non-overlapping" primer. The "n" and "o" assays shared the same opposite primer (SEQ ID NO: 3), and probe (SEQ ID NO: 4), and used artificial template SEQ ID NO: 5. The hairpin assays in 7(b) used the same dual-labeled primer (SEQ ID NO: 6) and artificial template (SEQ ID NO: 9). In Fig. 7(b), the six amplification curves correspond to three dilutions (10³, 10⁴ and 10⁵ copies) and "overlap" ("o"; corresponding to curves 730', 740' and 750', respectively) and "non-overlap" ("n"; corresponding to curves 730, 740 and 750, respectively) assay designs. The amplification curves for "overlap" assays were steeper and plateaued at a higher signal than "non-overlap" curves. All assays used primers and probes at concentrations of 300 nM and were performed in 10 reaction volume using Life Technologies Gene Expression Master mix on a Roche LightCycler480.

It is to be understood that this disclosure is not limited to specific compositions, method steps, as such can 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. Methods recited herein can be carried out in any order of the recited events that is logically possible, as well as the recited order of events. Also, it is contemplated that any optional feature of the disclosed variations described can be set forth and claimed independently, or in combination with any one or more of the features described herein.

All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques which are within the skill of the art. Such techniques are explained fully in the literature.

Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present disclosure. Various methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The section headings herein shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Further, a description of a technology in the "Background" is not to be construed as an admission that the technology is prior art to any invention(s) in this disclosure. Neither are the "Summary" or "Abstract" to be considered as a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to "invention" in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the inventions(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not constrained by the headings set forth herein.

Definitions

A "target nucleic acid" or "target polynucleotide" is a nucleic acid sequence of interest to be detected potentially present in a sample prior to any changes in the nucleic acid sequence composition made during sample processing, and may be either single- stranded or double stranded.

As used herein, the term "primer" refers to a polynucleotide that is serving as an initiation site for polymerase extension during PCR. In some embodiments, primers can have one or several labels and are called "primer-probes". A "5 '-probe portion" of a primer- probe includes at least one label that is used to generate a detectable signal.

A "digestion-driving primer" is a primer that drives the 5 'nuclease cleavage activity of a polymerase during PCR.

A "universal tag" is a part of the primer or amplicon with an artificial sequence that does not match the target nucleic acid, with the possible exception of very short tags that either intentionally or accidentally match target nucleic acid.

"Primer extension" is the enzymatic addition, i.e. polymerization, of monomeric nucleotide units to a primer while the primer is hybridized (annealed) to a template polynucleotide.

An "amplicon" is a DNA sequence generated in an amplification reaction.

Amplicons may include both target DNA sequences, and universal tagging sequences.

A "probe" is an oligonucleotide that carries at least one label that is used to generate a detectable signal. Probes usually have a blocked 3' end to prevent their extension by a polymerase.

A "universal tag" is a part of the primer, probe or amplicon (after primer extends) comprising an artificial sequence that does not match the target nucleic acid, with the possible exception of very short tags that either intentionally or accidentally match the target nucleic acid.

"Primer extension" is the enzymatic addition, i.e. polymerization, of monomeric nucleotide units to a primer while the primer is hybridized (annealed) to a template polynucleotide.

An "amplicon" is a DNA sequence generated in PCR reaction. Amplicons may include both target DNA sequences, and universal tagging sequences.

"qPCR" refers to quantitative Polymerase Chain Reaction, where the signal is measured during and/or at the end of PCR.

"C_t" (also referred to as C_p and C_q) is defined as the cycle at which fluorescence is determined to be statistically significant above background. The threshold cycle is inversely proportional to the log of the initial copy number. The more target molecules are present to begin with, the fewer the number of cycles it takes to get to a point where the fluorescent signal is detectable above background.

A "universal primer/probe" is a primer and/or probe that has one or several universal tags that do not match the target nucleic acid and may optionally contain a short stretch of target nucleic acid that occurs at the locus that is detected.

A "5 '-flap" is a universal tag at the 5' end of a primer, probe or amplicon. In some embodiments, it is cleavable by an enzyme with 5 ' nuclease activity.

The term "allele," as used herein, means a particular genetic variant or

polymorphism in the sequence of a gene, representing an alternative form of the gene.

As used herein, the phrase "allele-specific PCR amplification" refers to

amplification using the polymerase chain reaction to amplify predominantly one allele.

The term "allele-specific," when used in reference to probes or primers, means that a particular position of the probe or primer is complementary to an allele of a target polynucleotide sequence in the strand this primer or probe is to anneal.

A "surface attached oligonucleotide (primer or probe"), often called "oligo on solid support," indicates that the oligonucleotide does not diffuse in the reactor volume, but stays at a certain predefined location. Examples include flat surfaces, e.g., microarrays, beads, blots on paper, gel, etc.

An "encoding or linking reaction" is a step of target nucleic acid detection that generates "encoded DNA molecules with universal tags" that correspond to target nucleic acids. PCR pre-amplification with 5' tailed (tagged) primers is an example of an encoding or linking reaction. An "encoded target" is target DNA with attached universal tags after the encoding reaction.

The term "wild-type," as used herein, refers to a gene or gene product that has the characteristics of that gene or gene product that is most frequently observed in a population and is, thus, arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant", as used herein, refers to a gene or gene product that displays modifications in sequence when compared to the wild-type gene or gene product.

Claims

What is claimed is:

1. A 5 'nuclease-based PCR method that detects the presence or measures the amount of a target or encoded target nucleic acid, the method comprising:

forming an amplicon,

annealing a probe comprising a 5 ' portion, and a digestion-driving primer comprising a 3 ' end, to the amplicon,

wherein the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the probe by at least one nucleotide when both are annealed to the amplicon

so that a 5 'nuclease reaction can precede the primer extension.

2. The method of claim 1 , wherein the digestion-driving primer and the probe comprise two parts of a single molecule, wherein the single molecule forms a circular structure when the two parts anneal to the amplicon.

3. The method of claim 1 , comprising:

providing a second primer with a 5 '-probe portion;

extending the second primer to form the amplicon, wherein said amplicon comprises a region complementary to the 5' - probe portion;

said amplicon forming an intramolecular hairpin stem structure during PCR by annealing of the 5 '-probe portion and said region complementary to the 5 '-probe portion; and, annealing the digestion-driving primer to the amplicon, so that the 3 ' end of the digestion- driving primer overlaps the 5 '-portion of the stem by at least one nucleotide.

4. A PCR reaction mixture comprising: (i) an enzyme having 5 'nuclease activity; (ii) a digestion-driving primer comprising a 3 ' end, and (iii) a probe comprising a 5 ' portion, wherein the 3 ' end of the digestion-driving primer and the 5 ' portion of the probe overlap by at least one nucleotide when both the primer and the probe are annealed to an amplicon formed in said mixture.

5. The reaction mixture of claim 4, wherein the digestion-driving primer and the probe comprise two parts of a single molecule, wherein the single molecule is capable of forming a circular structure when the two parts anneal to the amplicon.

6. The reaction mixture of claim 4 further comprising:

a second primer comprising a 5 '-probe portion, said second primer capable of generating an amplicon having a region complementary to the 5 ' probe portion;

wherein said amplicon is capable of forming an intramolecular hairpin stem structure by annealing of the 5 '-probe portion and the region complementary to the 5 '-probe portion; wherein the digestion-driving primer can anneal to said amplicon, so that the 3 ' end of the digestion-driving primer overlaps the 5 ' portion of the stem.

7. The reaction mixture of claim 4, 5, or 6 wherein the 5' portion of the probe when annealed to the amplicon is more thermodynamically stable as compared to the 3 ' region of the digestion-driving primer when it is annealed to the amplicon.

8. The reaction mixture of claim 7, wherein modified nucleotides are used to alter the thermodynamic stability of the 3 ' region of the digestion driving primer and the 5 ' portion of the probe when the primer and probe are annealed to the amplicon.

9. The reaction mixture of claim 7 wherein the 3' end of the primer has a mismatch (non- Watson-Crick pair) or blocked non-extendable 3 ' end when annealed to the amplicon.

10. The reaction mixture of claim 4, 5, or 6 wherein the (a) probe, (b) primer-probe after extension or (c) digestion-driving primer comprise a label and label quencher that are capable of being separated from each other by said 5 'nuclease reaction and generating a detectable signal that allows detection of the presence and/or the amount of target nucleic acid.

11. The reaction mixture of claims 4, 5, 6 wherein the 5' portion of the probe comprises a flap sequence tag comprising a label quencher or a label configured to generate a fluorescent or electrochemical signal when the label quencher or the label are cleaved off together with the flap.

12. A software tool comprising: instructions to design primers and probes for methods according to claims 1-3 or for preparing mixtures according to claims 4-6, said instructions comprising choosing a probe and a digestion-driving primer that match a target nucleotide sequence, or tags in an encoded target sequence, so that there is an overlap of at least one nucleotide between the 5 ' portion of the probe and the 3 ' end of the digestion-driving primer.

13. A kit comprising a digestion-driving primer, second primer and a probe said probe can optionally be covalently connected of either of the two primers for use in mixtures according to claims 4-6, wherein all primers and probes required for a specific application are delivered to customers mixed together or separately and primers and probes can be inventoried, pre-designed or designed when customers submit their target sequences.

14. A method to increase specificity of mutation-specific PCR, the method comprising: providing a template comprising a putative mutated allele;

annealing to said template a blocking oligo having a 5 ' base which matches the mutated allele; and

annealing to said template a mutation-specific primer having a 3 ' end which matches said mutated allele;

wherein a 5 'nuclease reaction cleaves the 5 ' end of the blocking oligo when the mutation- specific primer and the oligo are annealed to the mutated template,

but wherein the blocking oligo is not effectively cleaved if the template is wild type, thereby further slowing mutation-specific primer extension on the wild-type template.

15. A PCR reaction mixture used to detect SNPs or mutations comprising:

(i) a polymerase with 5 'nuclease activity;

(ii) a mutation-specific primer;

(iii) a blocking oligo with blocked or otherwise un-extendable 3 ' end; and

(iv) template comprising a putative mutated allele,

such that the 3 ' end of the primer and at the 5 ' end of the blocking oligo when both are annealed to the template match the mutated allele, and

wherein the 5 'nuclease activity is capable of cleaving the 5' end of the blocking

oligonucleotide, so that the primer can extend on a mutant allele, but the 5 'nuclease cleavage is substantially reduced if the template is wild-type, thus providing additional blocking for primer extension.

16. The mixture of claim 15 wherein the mutation- specific primer and blocking

oligonucleotide comprise a single molecule that forms a circular structure when annealed to the template.

17. The reaction mixture of claim 4, 5 or 6, wherein the digestion-driving primer, probe and/or amplicon (after surface-bound primer extends) and optionally the second primer are attached to a surface during PCR or a bridge amplification and contain at least one label and optionally least one label quencher.