Amplification and data collection protocol for rapid genotyping

Abstract

Disclosed herein are methods of genotyping a crude nucleic acid sample, the methods including: subjecting the crude nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the crude nucleic acid sample from a source; performing rapid PCR on the crude nucleic acid sample by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle; and analyzing results from the rapid PCR to determine the nucleic acid sample's genotype. Each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data. The at least second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data. Methods of detecting a disease in a subject are also disclosed.

Description

AMPLIFICATION AND DATA COLLECTION PROTOCOL FOR RAPIDGENOTYPINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 553,998, titled “Amplification and Data Collection Protocol for Rapid Genotyping,” filed February 15, 2024, the entirety of which is incorporated herein by this reference.FIELD

[0002] The present disclosure relates to amplification and data collection protocol s / methods for rapid genotyping a crude nucleic acid sample directly from a source. More specifically, amplification and data collection protocols / methods that allow for rapid amplification, detection, and / or quantitation of nucleic acid molecules in a crude nucleic acid sample.BACKGROUND INFORMATION

[0003] Genetic variation accounts for a wide variety of differences among people, such as eye color and blood group. Genetic variation also impacts the likelihood that a person will develop or succumb to a particular disease and whether a person is likely to have a favorable or adverse response to a particular drug. Indeed, single gene differences in individuals have been associated with elevated risk for acquiring a variety of diseases, such as cystic fibrosis and sickle cell disease. More complex interrelationships among multiple genes and, in some circumstances, the environment can be responsible for increased risk of some common diseases, such asdiabetes, cancer, stroke, Alzheimer's disease, Parkinson's disease, depression, alcoholism, heart disease, arthritis and asthma.

[0004] To assist in the understanding of which genetic variations impose a higher risk of disease development and / or poor drug efficacy, genetic-based research tools and analytical methods have been developed. In recent years, the polymerase chain reaction (PCR) has become a method of choice for detecting genetic variation among individuals. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region(s) of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. PCR is capable of producing a selective enrichment of a specific DNA sequence by, e.g, a factor of 109(see, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188, and Saiki et al., 1985, Science 230: 1350).

[0005] When PCR was first popularized in the late 1980s, the process was slow. A typical protocol was one minute for denaturation at 94° C., two minutes for annealing at 55° C., and three minutes for extension at 72° C. When the time for transition between temperatures was included, 8 minute cycles were typical and completion of 30 cycles took about four hours.

[0006] Currently, PCR procedures involve obtaining a nucleic acid sample and subjecting the sample to PCR preparation procedures (e.g., mixing with a lysis buffer or a DNA extraction solution or a precipitating agent, and / or diluting) before starting the PCR.Additionally, current PCR procedures require the use of specialized equipment (e.g., centrifuges and filters) for the purification and / or preparation of the nucleic acid sample. Because current PCR procedures require preparation of the sample beforehand, these procedures can take anywhere from about 1 hour (e.g., at least 40 mins) to weeks to complete, depending on the amount of sample and the type of PCR performed on the sample. These requirements of current PCR procedures have hindered their ability to perform rapid genotyping outside of a specialized lab infrastructure, e.g., in a mobile or field setting. Extraction and amplification alone can take hours, if not days, depending on the type of organism, the length of the nucleic acid strand, andthe number of cycles. In addition, commercially available devices for performing PCR procedures require skilled labor, running water, and electricity, all of which can be absent in impoverished communities experiencing a disease outbreak.

[0007] Thus, to address the foregoing issues, the present disclosure offers a more economically friendly and rapid process for genotyping nucleic acid samples.SUMMARY

[0008] Disclosed herein is a method of genotyping a crude nucleic acid sample, the method comprising: subjecting the crude nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the crude nucleic acid sample from a source; performing rapid PCR on the crude nucleic acid sample by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and analyzing results from the rapid PCR to determine a genotype of the crude nucleic acid sample.

[0009] In the aforementioned method, the rapid PCR can be performed in less than 30 minutes.

[0010] In the aforementioned method, the rapid PCR can be performed in about 20 minutes.

[0011] In the aforementioned method, the performing rapid PCR can further comprise: incubating the crude nucleic acid sample in the PCR mixture for a set period of time, performing a pre-PCR read of the incubated crude nucleic acid sample for about 10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post -PCR read for about 10 seconds of ampliconsgenerated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

[0012] In the aforementioned method, the first plurality of amplification cycles includes four amplification cycles where each cycle includes performing the second denaturation for about one second followed by annealing and extension for 3 seconds, and the at least one second amplification cycle includes performing the second denaturation for about one second followed by annealing and extension for 10 seconds.

[0013] In the aforementioned method, the performing rapid PCR on the crude nucleic acid sample can include subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle 8 times.

[0014] In the aforementioned method, the pre-PCR read and the post-PCR read can be performed at a temperature from 55°C to 65°C.

[0015] In the aforementioned method, the initial denaturation and the second denaturation can be performed at a temperature from 85°C to 100°C.

[0016] In the aforementioned method, the pre-PCR read and the post-PCR read can be performed at a temperature of about 60°C, and the initial denaturation and the second denaturation are performed at a temperature of about 95°C.

[0017] Disclosed is a method of detecting a disease in a subject, comprising: subjecting a reaction mixture consisting essentially of a polymerase chain reaction (PCR) mixture and a crude nucleic acid sample from the subject to rapid PCR by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least one second amplification cycle includes performing denaturation followed by annealing andextension while simultaneously collecting amplification data; and determining a presence and / or amount of at least one target nucleic acid sequence in the crude nucleic acid sample, the at least one target nucleic acid sequence being a biomarker of the disease.

[0018] In the aforementioned methods, the PCR mixture can include a hot-start component.

[0019] In the aforementioned methods, the hot-start component can inhibit polymerase activity of a polymerase in the PCR mixture and can include at least one or more of an oligonucleotide, a temperature-dependent ligand, an aptamer, and antibodies specific to the polymerase and / or an agent that mediates a reversible chemical modification of the polymerase.

[0020] In the aforementioned methods, the method can be performed in the absence of a lysing solution and / or a nucleic acid extraction solution.

[0021] In the aforementioned methods, the source can be a buccal cavity.

[0022] In the aforementioned methods, the crude nucleic acid sample can be obtained using a swab, the swab being at least one selected from a dry swab, a dry foam swab, a flocked swab and a moisten or wet swab.

[0023] In the aforementioned methods, the swab can be formed of foam, cotton, flock, rayon, polyester, calcium alginate or any combination thereof.

[0024] In the aforementioned methods, the swab is not subjected to a transport medium prior to the crude nucleic acid sample being subjected to the PCR mixture, or in the reaction mixture.

[0025] In the aforementioned methods, the source can be a buccal cavity, and the method further comprises swabbing the buccal cavity to obtain the crude nucleic acid sample.

[0026] In the aforementioned methods, the source can be a hair follicle.

[0027] In the aforementioned methods, the crude nucleic acid sample can be obtained at least 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.

[0028] In the aforementioned methods, the crude nucleic acid sample can be obtained within 24 to 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.

[0029] In the aforementioned methods, the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, can be an undiluted sample.

[0030] In the aforementioned methods, the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, can be an unpurified sample.

[0031] In the aforementioned methods, the subjecting of the reaction mixture to the rapid PCR can include performing one selected from polymerase chain reaction (PCR), real-time PCR, quantitative PCR, multiplex PCR, methylation-specific PCR and endpoint PCR.

[0032] In the aforementioned methods, the subjecting of the reaction mixture to the rapid PCR can include: incubating the crude nucleic acid sample in the PCR mixture for a set period of time, performing a pre-PCR read of the incubated crude nucleic acid sample for about 10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

[0033] In the aforementioned methods, the post-PCR read can be an end-point PCR read.

[0034] In the aforementioned methods, the PCR mixture or the reaction mixture can contain deoxynucleotide triphosphates, magnesium ions, potassium ions, a buffer solution, at least one primer pair specific for nucleotides in a target nucleic acid in the crude nucleic acidsample, at least one probe specific for nucleotides in the target nucleic acid, and at least one thermostable polymerase, wherein the at least one probe includes a detectable label.

[0035] In the aforementioned methods, the target nucleic acid can be a biomarker for a disease.

[0036] In the aforementioned methods, the at least one thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, Platinum Taq DNA polymerase, Platinum II Taq Hot-Start DNA polymerase, AmpliTaq DNA polymerase, Invitrogen Taq DNA, Dream Taq DNA polymerase and mutants or derivatives thereof having DNA polymerase activity.

[0037] In the aforementioned methods, the PCR mixture can contain a PCR inhibitor blocking agent.

[0038] In the aforementioned methods, the PCR inhibitor blocking agent can be selected from the group consisting of an albumin, a gelatin, and a combination thereof.

[0039] In the aforementioned methods, the performing the rapid PCR, or the determining the presence and / or amount of at least one target nucleic acid sequence, can include using at least one probe and at least one reporter dye.

[0040] In the aforementioned methods, the method can be used to perform a genotyping assay.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0042] FIG. 1 depicts a flow diagram of an exemplary embodiment of the method for genotyping a nucleic acid sample.

[0043] FIG. 2 is a conventional workflow for sample preparation and PCR.

[0044] FIG. 3A depicts genotyping results of a nucleic acid sample prepared and amplified using a conventional method.

[0045] FIG. 3B depicts genotyping results of a crude nucleic acid sample obtained and directly amplified according to exemplary embodiments.DETAILED DESCRIPTION

[0046] The present disclosure relates to amplification and data collection protocol s / methods for rapid genotyping a crude nucleic acid sample directly from a source. More specifically, amplification and data collection protocols / methods that allow for rapid amplification, detection, and / or quantitation of nucleic acid molecules in a crude nucleic acid sample.

[0047] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books and treatises, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose.

[0048] While the present disclosure is described in conjunction with various embodiments, it is not intended that the present disclosure be limited to such embodiments. On the contrary, the present disclosure encompasses various alternatives, modifications and equivalents of the embodiments disclosed herein, as will be appreciated by those of skill in the art.

[0049] Most of the words used in this specification have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined in the specification have the meaning provided in the context of the present disclosure as a whole, and as aretypically understood by those skilled in the art. In the event that a conflict arises between an art- understood definition of a word or phrase and a definition of the word or phrase as specifically described in this specification, the specification shall control.

[0050] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes” and / or “including,” if used herein, specify the presence of stated features, steps, elements and / or components, but do not preclude the presence or addition of one or more other features, steps, elements, components and / or groups thereof.

[0051] As used herein, “polymerase chain reaction” or PCR is a an amplification of nucleic acid consisting of (i) an initial denaturation step which separates the strands of a double stranded nucleic acid sample, followed by repetition of (ii) an annealing step, which allows amplification primers to anneal specifically to positions flanking a target sequence; (iii) an extension step which extends the primers in a 5' to 3' direction thereby forming an amplicon polynucleotide complementary to the target sequence, and (iv) a denaturation step which causes the separation of the amplicon from the target sequence. Each of the above steps may be conducted at a different temperature, preferably using a thermocycler or an automated thermocycler ( such as QuantStudio 1 Real-time PCR System, QuantStudio 3 Real-Time PCR System, QuantStudio 3 Real-Time PCR System, QuantStudio 5 Real-Time PCR System, QuantStudio 5 Dx Real-Time PCR System, QuantStudio 5 Food Safety Real-Time PCR System, Pharmaceutical Analytics QuantStudio 5 Real-Time PCR System, QuantStudio 6 Pro Real-Time PCR System, QuantStudio 7 Pro Real-Time PCR System, QuantStudio 7 Flex Real-Time PCR System, QuantStudio 7 Pro Dx Real-Time PCR System, QuantStudio 12K Flex Real-Time PCR System, Automated Thermal Cycler, 7500 Fast Food Safety Real-Time PCR System, SimpliAmp Thermal Cycler, MiniAmp Thermal Cycler, ProFlex PCR System, or Veriti Pro Dx Thermal Cycler, all manufactured by Applied Biosystems LLC, a division of Life Technologies Corporation, a subsidiary of Thermo Fisher Scientific).

[0052] If desired, RNA samples can be converted to DNA / RNA heteroduplexes or to duplex cDNA by methods known to one of skill in the art.

[0053] The PCR method can also include real-time PCR, quantitative PCR, multiplex PCR, methylation-specific PCR or end-point PCR, reverse transcriptase-PCR and other reactions that follow principles of PCR. Exemplary PCR processes for amplifying nucleic acids is covered by U.S. Patent Nos. 4,683,195 and 4,683,202, which are herein incorporated in their entirety by reference for a description of the process. The reaction conditions for any PCR comprise the chemical components of the reaction and their concentrations, the temperatures used in the reaction cycles, the number of cycles of the reaction, and the durations of the stages of the reaction cycles.

[0054] As used herein, the term “target,” “target sequence,” “target template,” or “target nucleic acid sequence” refers to a nucleic acid sequence or region of a nucleic acid which is to be either amplified, detected, or both. Typically, in a PCR, the target sequence resides between the two primer sequences used for amplification.

[0055] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0056] As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleotide triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or whichaffect pH, ionic strength, etc ), and at a suitable temperature. The primer can be single-stranded for maximum efficiency in amplification. A primer can be l l bases or longer; more specifically, about 17 bases or longer, although shorter or longer primers can be used depending on the need. As will be appreciated by those skilled in the art, the oligonucleotides can be used as one or more primers in various extension, synthesis or amplification reactions.

[0057] The complement of a nucleic acid sequence, as used herein, refers to an oligonucleotide or a polynucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 31end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids can be included in the crude nucleic acid samples of the present disclosure and include, for example, inosine and 7- deazaguanine. Complementarity need not be perfect; stable duplexes can contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically by considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength, and incidence of mismatched base pairs. Stability of a nucleic acid duplex is measured by the melting temperature (“Tm”). The Tmof a particular nucleic acid duplex under specified conditions is the temperature at which half of the base pairs have disassociated.

[0058] "PCR-compatible" refers to any composition, solution, compound, reagent, etc. that is compatible with subsequent use in PCR assays and is relatively non-inhibitory of the enzymatic polymerase chain reaction. PCR-compatible products demonstrate relatively minimal or no inhibition of PCR amplification, as evidenced by comparison of PCR results with the relevant positive and negative controls. PCR assays can include, but are not limited to, DNA genotyping systems, TaqMan™ or SYBR™ green real-time PCR assays for DNA quantification, multiplex PCR assays including those designed to genotype short tandem repeats, single nucleotide polymorphism (SNP), etc.

[0059] As used herein, "amplify" refers to the process of enzymatically increasing the amount of a specific nucleotide sequence. This amplification is not limited to but is generally accomplished by PCR. As used herein, the term “amplifying” refers to any means by which at least a part of a target polynucleotide, target polynucleotide surrogate, or combinations thereof, isreproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Any known method can be used to amplify the target polynucleotide. Any in vitro means for multiplying the copies of a target sequence of nucleic acid can be utilized. These include linear, logarithmic, or any other amplification methods. Exemplary methods include polymerase chain reaction (PCR), partial destruction of primer molecules (see, e.g., PCT Application Pub WO 2006 / 087574), ligase chain reaction (see, e.g., Wu, et al. Genomics 4:560-569 (1990) and Barany, et al. Proc. Natl. Acad. Sci. USA 88: 189-193 (1991), Q0 RNA replicase systems (see, e.g., WO 1994 / 016108), RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; Lizardi, et al. Nat. Genet. 19:225-232 (1998); and Baner, et al. Nucleic Acid Res. 26: 5073-5078 (1998)), and strand displacement amplification (SDA) (Little, et al. Clin. Chem. 45:777-784 (1999)), among others. Many systems are suitable for use in amplifying target nucleic acids and are contemplated herein as would be understood by one of skill in the art. In exemplary embodiments of the methods disclosed herein, nucleic acid amplification reactions are performed by a PCR. In exemplary embodiments, the PCR is a quantitative PCR (qPCR). In exemplary embodiments, the qPCR is performed by real time PCR. In exemplary embodiments, the PCR is an endpoint PCR. In exemplary embodiments, the PCR is multiplex PCR. In exemplary embodiments, the PCR is methylation-specific PCR. In exemplary embodiments, the PCR involves thermal cycling. In exemplary embodiments, the thermal cycling is programmed for fast or rapid thermal cycling.

[0060] As used herein the terms “annealing” and “hybridization” are used interchangeably and mean the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure. In exemplary embodiments, the primary interaction is base specific, e.g., A / T and G / C, by Watson / Crick and Hoogsteen-type hydrogen bonding. In exemplary embodiments, base-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for hybridizing nucleic acid probes and primers to complementary and substantially complementary target sequences are well known, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J. Wetmur and N. Davidson, Mol. Biol. 31 :349 et seq. (1968). Annealing is influenced by, among other things, the length of the probes and the complementary targetsequences, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, annealing conditions will depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation. Further, probes and primers can be designed to be complementary to a target sequence, such that hybridization of the target and the probes or primers occurs. It will be appreciated, however, that this complementarity need not be perfect; there can be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids. However, if the number of base pair mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes or primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions.

[0061] The term “label” as used herein refers to any atom or molecule which can be used to provide a detectable signal, and which can be attached to a nucleic acid or protein. Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, and / or enzymatic activity. In exemplary embodiments, the detectable signal is a quantifiable signal.

[0062] The terms “amplification cycle” and “PCR cycle” are used interchangeably herein and as used herein refers to the denaturing of a double-stranded polynucleotide sequence followed by annealing of a primer sequence to its complementary sequence and extension of the primer sequence.

[0063] The terms “amplicon,” “amplification product” and “amplified sequence” are used interchangeably herein and refer to a broad range of techniques for increasing polynucleotide sequences, either linearly or exponentially and can be the product of an amplification reaction. An amplicon can be double-stranded or single-stranded, and can include the separated component strands obtained by denaturing a double-stranded amplification product. In exemplary embodiments, the amplicon of one amplification cycle can serve as atemplate in a subsequent amplification cycle. Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step. Other nonlimiting examples of amplification include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR). Amplification methods can include thermal -cycling or can be performed isothermally. In various embodiments, the term “amplification product” and “amplified sequence” includes products from any number of cycles of amplification reactions.

[0064] As used herein, the terms “amplification primer” and “oligonucleotide primer” are used interchangeably and refer to an oligonucleotide, capable of annealing to an RNA or DNA region. The region annealed to can be adjacent a target sequence, including but not limited to a SNP, a STR or mutation region, and serving as an initiation primer for DNA synthesis under suitable conditions well known in the art. Typically, a PCR reaction employs an “amplification primer pair” also referred to as an “oligonucleotide primer pair” including an “upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified. A first primer and a second primer may be either a forward or reverse primer respectively, and are used interchangeably herein and are not to be limiting.

[0065] As used herein, “extension” refers to the amplification cycle after the primer oligonucleotide and target nucleic acid have annealed to one another, wherein the polymerase enzyme catalyzes primer extension, thereby enabling amplification, using the target nucleic acid as a replication template.

[0066] As used herein, the terms "upstream" and "downstream" are used in relation to the synthesis of the nascent strand that is primed by a target-specific primer. Thus, for example, a target-specific probe hybridized to a region of the target nucleic acid that is "downstream" of the region of the target nucleic acid to which the primer is hybridized is located 3 ' of the primer and will be in the path of a polymerase extending the primer in a 5' to 3' direction.

[0067] As used herein, “nucleotide” refers to a base-sugar-phosphate combination. A “nucleoside” refers to a base-sugar combination. Nucleotides are monomeric units of a nucleic acid sequence (e.g., DNA and RNA). The term nucleotide includes mono-, di- and triphosphate forms of deoxyribonucleosides and ribonucleosides and their derivatives. dNTPs can be unlabeled, or they can be detectably labeled by coupling them by methods known in the art withradioisotopes (e.g, H3, C14, P32 or S35), vitamins e.g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels and dioxigenin. Labeled dNTPs can be obtained commercially, for example from Thermo Fisher Scientific or Sigma-Aldrich Company.

[0068] As used herein, “polynucleotide” and “oligonucleotide” refer to a synthetic or biologically produced molecule comprising a covalently linked sequence of nucleotides which can be joined by a phosphodiester bond between the 3' position of the pentose of one nucleotide and the 5' position of the pentose of the adjacent nucleotide. In addition, a polynucleotide or oligonucleotide can contain modified or non-naturally occurring sugar residues (e.g., arabinose) and / or modified base residues. A polynucleotide or oligonucleotide can also comprise blocking groups that prevent the interaction of the molecule with particular proteins, enzymes or substrates.

[0069] As used herein, "nucleic acid" refers to the nucleic acid molecule or molecules, DNA or RNA (ribonucleic acid) in any form. "DNA" refers to deoxyribonucleic acid in its various forms as understood in the art, such as genomic DNA, cDNA, isolated nucleic acid molecules, vector DNA, and chromosomal DNA. As used herein, the term "isolated nucleic acid molecule" or "isolated nucleic acid" refers to a nucleic acid molecule (DNA or RNA of any form) that has been removed from its native environment. Some examples of isolated nucleic acid molecules are recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, nucleic acids obtained from forensic and other samples comprising biological material, such as blood, semen, saliva, skin tissue, etc., and synthetic DNA molecules. An "isolated" nucleic acid can be free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5’ and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.

[0070] As used herein, a “biological sample” can include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include buccal samples, blood and blood fractions or products (e.g., serum, platelets, red blood cells, and the like), lymph, bone marrow, sputum, bronchoalveolar lavage, amniotic fluid, hair, skin, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, etc. Biological samples can also be fresh, frozen or formalin- or paraformalin-fixed paraffin- embedded tissue (FFPE).

[0071] As used herein, the term “crude nucleic acid” refers to a specimen of biological origin containing nucleic acids, which has not undergone procedures for the isolation, disintegration, extraction, dilution or purification of those nucleic acids. Crude samples can include, but are not limited to, blood, diluted blood, blood on paper, buccal swabs, and buccal swabs on a substrate for samples storage, such as FTA paper. One of skill in the art will recognize an enormous variety of other crude samples that can be used in the disclosed methods, including those not specifically disclosed herein.

[0072] As used herein the term “Ct” or “Ct value” refers to threshold cycle and signifies the cycle of a PCR amplification assay in which signal from a reporter that is indicative of amplicon generation (e.g., fluorescence) first becomes detectable above a background level. In exemplary embodiments, the threshold cycle or “Ct” is the cycle number at which PCR amplification becomes exponential. In exemplary embodiments, the signal from a reporter, such as fluorescence, is described as delta Rn. As used herein, the term “dRn” or “delta Rn” refers to the difference in the normalized reporter signal (Rn) subtracted from the background signal (baseline) which is then normalized by a passive reference signal. Delta Rn can be determined by the formula Rn - Rn , where Rn+is the Rn value for a reaction involving all components, including the template, and Rn is the value for an unreacted sample.

[0073] As used herein, “a lysing solution” refers to a solution or a mixture formulated to enable lysis, or breaking open, of cells in a biological sample. Examples of a lysis solution include a nonidet P-40 (NP-40) solution, a sodium dodecyl sulfate (SDS) solution, an ammonium-chlori de-potassium (ACK) buffer, a Radiolmmuno Precipitation assay (RIPA)solution, a cell lysis solution, and immunoprecipitation (IP) solution. However, embodiments are not limited thereto.

[0074] As used herein, “a nucleic acid extraction solution” is a solution or mixture formulated to enable extraction, isolation and / or purification of nucleic acid from a biological sample. Examples of a nucleic acid extraction solution include a phenol -chloroform buffer, a chaotropic agent (e.g., a guanidine or guanidine isothiocyanate solution). However, embodiments are not limited thereto.

[0075] One aspect of the present disclosure is a method for genotyping a nucleic acid sample, the method including at least one or more of: subjecting a crude nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the crude nucleic acid sample from a source; performing rapid PCR on the crude nucleic acid sample by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and analyzing results from the rapid PCR to determine the nucleic acid sample’s genotype.

[0076] FIG. 1 depicts an exemplary embodiment of the method wherein (i) the crude nucleic acid sample is obtained from the source (such as a buccal cavity), (ii) the crude nucleic acid sample is directly subjected to a PCR mixture without any intervening step and the crude nucleic acid is left to incubate in the PCR mixture for a set period of time, and (iii) the incubated crude sample is subjected to a rapid PCR.

[0077] In exemplary embodiments, the method includes obtaining the crude nucleic acid sample from a source, wherein the source is selected from one or more of blood, semen, saliva, hair follicles, skin tissue, mucus or any combination thereof. In other exemplary embodiments, the source is selected from a virus, an archaea, a protist, a prokaryote or an eukaryote.

[0078] In exemplary embodiments, the crude nucleic acid sample exists within its normal cellular, bacterial, or viral environment.

[0079] In exemplary embodiment, the crude nucleic acid sample is composed of living cells. In other exemplary embodiment, the crude nucleic acid sample is composed of dead cells. In yet other exemplary embodiment, the crude nucleic acid sample is composed of a combination of living cells and dead cells.

[0080] In exemplary embodiments, the crude nucleic acid sample contains a target nucleic acid sequence. The target nucleic acid sequence can be DNA, , cell-free DNA (cfDNA), nuclear DNA (nDNA), mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid DNA, environmental DNA (eDNA), relaxed circular (rcDNA), covalently closed circular DNA (cccDNA), circulating tumor DNA (ctDNA), RNA, messenger RNA (mRNA), transfer RNA (tRNA), small interfering RNA (siRNA), microRNA (miRNA), ribosomal RNA (rRNA), piwi- interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) or other mature small RNA, and can include nucleic acid analogs or other nucleic acid mimics. The target nucleic acid sequence can be methylated, non-methylated, or both. The target nucleic acid sequence can be bisulfite-treated and have non-methylated cytosines converted to uracil.Further, it will be appreciated that “target nucleic acid” can refer to the target nucleic acid itself, as well as surrogates thereof, for example, amplification products and native sequences.

[0081] The crude nucleic acid samples can be obtained from any number of biological sources, including without limitation, viruses, archae, protists, prokaryotes and eukaryotes, for example, from a biological sample obtained from a eukaryotic organism, such as a mammal e.g., a chimpanzee or a human; a cow; a dog; a cat; a rodent, e.g., a guinea pig, a rat, a mouse; a rabbit; or a bird; a reptile; or a fish. The crude nucleic acid sample can be obtained from cells, tissues, organs, or organisms in different developmental stages. The crude nucleic acid samples can also be obtained from cancer cells and precancerous cells obtained from animals, including humans. The crude nucleic acid sample can also be obtained from cell culture lines, including transformed and non-transformed cell culture lines. Crude nucleic acid samples can be obtained from a variety of sources. These include, but are not limited to, for example clothing, soil, paper, metal surfaces, air, water, plant parts, as well as human and / or animal skin, hair, blood, serum, feces, milk, saliva, urine, and / or other secretory fluids.

[0082] In exemplary embodiments, the crude nucleic acid sample is obtained using a swab, the swab being at least one selected from a dry swab, a dry foam swab, a flocked swab and a moisten or wet swab. In exemplary embodiments, the swab is formed from foam, cotton, flock, rayon, polyester, calcium alginate or any combination thereof. In exemplary embodiments, the swab is not subjected to a transport medium prior to being subjected to the PCR mixture.

[0083] In exemplary embodiments, the source of the crude nucleic acid sample is a buccal cavity, and the method further comprises swabbing the buccal cavity to obtain the crude nucleic acid sample.

[0084] In exemplary embodiments, the source of the crude nucleic acid sample is a hair follicle.

[0085] In exemplary embodiments, the crude nucleic acid sample is obtained at least 120 hours prior to being subjected to the PCR mixture. In exemplary embodiments, the crude nucleic acid sample is obtained within 24 to 120 hours prior to being subjected to the PCR mixture.

[0086] In exemplary embodiments, the crude nucleic acid sample subjected to the PCR mixture is an undiluted sample. For instance, an undiluted crude nucleic acid sample is absent of solvent or lysis buffer other than solutions that exist within its normal cellular, bacterial, or viral environment. In exemplary embodiments, the crude nucleic acid sample subjected to the PCR mixture is an unpurified sample. For instance, an unpurified sample has not been subjected to physical methods (such as filtration or purification), and / or chemical methods (such as lysed), of extraction.

[0087] In exemplary embodiments, the method includes subjecting the nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the sample, wherein the PCR mixture contains at least one or more of deoxynucleotide triphosphates (dNTPs), magnesium ions, potassium ions, a buffer solution, a forward primer, a reverse primer and at least one DNA polymerase. In other exemplary embodiments, the PCR mixture contains a DNA polymerase, a salt, an antifoam agent, and a combination of a dNTP and a dNTP derivative.

[0088] In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase selected from those listed below. In other exemplary embodiments, the thermostable DNA polymerase is a Taq-derived DNA polymerase.

[0089] When referring to a thermostable DNA polymerase, one unit of activity is the amount of enzyme that will incorporate 10 nanomoles of dNTPs into acid-insoluble material (i.e., DNA or RNA) in 30 minutes under standard primed DNA synthesis conditions.

[0090] In exemplary embodiments, the PCR mixture includes more than one polymerase. Such polymerases can be any enzyme capable of replicating a DNA molecule. In exemplary embodiments, the PCR mixtures include a DNA-dependent DNA polymerase, an enzyme for reverse transcription (RNA-dependent DNA polymerase), and / or a combination of both types of enzymes. In exemplary embodiments, a combination of DNA dependent DNA polymerases and / or a combination of RNA-dependent DNA polymerase are present in the PCR mixtures disclosed herein.

[0091] In exemplary embodiments, the polymerases used in the PCR mixtures disclosed herein are thermostable DNA polymerases. In exemplary embodiments, the thermostable DNA polymerases as used herein are not irreversibly inactivated when subjected to elevated temperatures for the time necessary to effect destabilization of single-stranded nucleic acids or denaturation of double-stranded nucleic acids during nucleic acid synthesis or PCR amplification. Irreversible denaturation of the enzyme refers to substantial loss of enzyme activity. In exemplary embodiments, a thermostable DNA polymerase that does not irreversibly denature at about 90°-100°C under conditions required for PCR amplification is included in the PCR mixture.

[0092] DNA polymerases disclosed herein can be isolated from natural or recombinant sources, by techniques that are well-known in the art (see, e.g., PCT Publication Nos. WO 92 / 06200; WO 96 / 10640; U.S. Patent Nos. 5,455,170; 5,912,155; and 5,466,591, the disclosures of which are fully incorporated herein by reference in their entireties), from a variety of thermophilic bacteria that are available commercially (for example, from American Type Culture Collection, Rockville, Md.) or can be obtained by recombinant DNA techniques (see, e.g., PCT Publication No. WO 96 / 10640 and U.S. Patent No. 5,912,155). Suitable for use as sources ofthermostable polymerases or the genes thereof for expression in recombinant systems are, for example, the thermophilic bacteria Thermits thermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosh and other species of the Pyrococcus genus, Bacillus sterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus, and Methanobacterium thermoautotrophicum, and mutants, variants or derivatives thereof.

[0093] In exemplary embodiments, the PCR mixtures provided herein include DNA polymerases for amplification and / or sequencing, wherein the DNA polymerases are selected from Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEP VENT™ DNA polymerase, Pyrococcus woos (Pwo) DNA polymerase, Pyrococcus sp KOD2 (KOD) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bea) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, Mycobacterium DNA polymerase (Mtb, Mlep), E. coli pol I DNA polymerase, a Stoffel fragment DNA polymerase, KI enow fragment, T5 DNA polymerase, T7 DNA polymerase, and generally pol I type DNA polymerases; mutants, variants and derivatives thereof, and combinations of the foregoing.

[0094] In exemplary embodiments, the PCR mixtures provided herein include DNA polymerases that are mesophilic, thermophilic, and / or thermophilic and thermostable. As used herein, the term “thermostable nucleic acid polymerase” refers to an enzyme which is relatively stable to heat when compared, for example, to nucleotide polymerases from E. coli and which catalyzes the polymerization of nucleotide triphosphates. Generally, the enzyme will initiate synthesis at the 3'-end of the primer annealed to the target sequence, and will proceed in the 5'- direction along the template, and if possessing a 5' to 3' nuclease activity, hydrolyzing intervening, annealed probe to release both labeled and unlabeled probe fragments, untilsynthesis terminates. A representative thermostable enzyme isolated from Thermus aquations (Taq) is described in U.S. Pat. No. 4, 889,818 and a method for using it in a PCR is described in Saiki etal., 1988, Science 239:487.

[0095] Suitable mesophilic DNA polymerases include Pol I family of DNA polymerases (and their respective Klenow fragments) any of which can be isolated from organism such as E. coli, H. influenzae, D. radiodurans, H. pylori, C. aurantiacus, R. prowazekii, T pallidum, Synechocystis sp., B. sublilis, L. lactis, S. pneumoniae, M. tuberculosis, M. leprae, M. smegmatis, Bacteriophage L5, phi-C31, T7, T3, T5, SP01, SP02, mitochondrial from S. cerevisiae MIP-1, and eukaryotic C. elegans, and / ). melanogaster (Astatke, M. et al., 1998, J Mol. Biol. 278, 147- 165), pol III type DNA polymerase isolated from any sources, and mutants, derivatives or variants thereof.

[0096] In exemplary embodiments, the polymerases used in the PCR mixtures disclosed herein have a 5’->3’ exonuclease activity. As defined herein, “5'— 3' nuclease activity” or “51to 3' nuclease activity” refers to that activity of a template-specific nucleic acid polymerase including either a 5'— >3' exonuclease activity traditionally associated with some DNA polymerases whereby nucleotides are removed from the 5' end of an oligonucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow fragment does not), or a 5'— >3' endonuclease activity wherein cleavage occurs more than one phosphodiester bond (nucleotide) from the 5' end, or both. For example, Taq DNA polymerase has a DNA synthesis-dependent, strand replacement 5'-3' exonuclease activity (see Gelfand, “Taq DNA Polymerase” in PCR Technology: Principles and Applications for DNA Amplification, Erlich, Ed. , Stockton Press, N.Y. (1989), Chapter 2). In exemplary embodiments, the PCR mixtures disclosed herein include an enzyme that has reverse transcriptase activity. Suitable enzymes having reverse transcriptase activity can be, for example, retroviral reverse transcriptases such as Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase, ASLV reverse transcriptases, as well as Lentivirus reverse transcriptases, or any corresponding mutants, variants or derivatives thereof having reverse transcriptase activity. As used herein, “mutants, variants, or derivatives” refer to all permutations of a chemical species, which mayexist or be produced, that still retains the definitive chemical activity of that chemical species. In exemplary embodiments, the PCR mixture includes at least one RNase H+ enzymes such as, for example, RNase H+ M-MLV or RNase H+ AMV reverse transcriptases. Alternatively, the reverse transcriptases can have reduced, substantially reduced, or eliminated RNase H activity (see, e.g., U.S. Pat. No. 7,078,208, the disclosure of which is fully incorporated by reference in its entirety). RNase H is a processive 5’ and 3’ ribonuclease that is specific for the RNA strand of RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). RNase H activity can be determined by a variety of assays, such as those described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992).

[0097] In exemplary embodiments, the polymerases used in the PCR mixtures disclosed herein have at least one reverse transcriptase possessing a mutation not found in naturally occurring reverse transcriptase. For example, the reverse transcriptase can be modified to contain a mutation that provides increased reverse transcriptase stability and / or functionality. Suitable enzymes can also include those in which terminal deoxynucleotidyl transferase (TdT) activity has been reduced, substantially reduced, or eliminated. Reverse transcriptases which exhibit such increased or decreased functionalities are described in, for example, U.S. Pat. Nos. 7,056,716 and 7,078,208.

[0098] In exemplary embodiments, the PCR mixtures provided herein include thermostable DNA polymerases selected from those listed above, mutants or derivatives thereof having DNA polymerase activity, and any combination of the foregoing. Taq DNA polymerase and mutant forms thereof are commercially available, for example, from Life Technologies (Carlsbad, CA), or can be isolated from their natural source, (e.g., from the thermophilic bacterium Thermus aquaticus for Taq polymerase), as described previously (see, e.g., U.S. Patent Nos. 4,889,818 and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties). Tne DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga neapolitana (see, e.g., PCT Publication No. WO 96 / 10640 and U.S. Patent No. 5,912,155), and Tma DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga maritima (see, e.g., U. S. Patent No. 5,374,553, the disclosure of which is incorporated herein by reference in its entirety). Exemplary thermostablepolymerases include, but are not limited to, AmpliTaq™ DNA polymerase, AmpliTaq™ Gold DNA polymerase, AmpliTaq™ Gold 360 DNA polymerase, DreamTaq™ DNA polymerase, Taq DNA polymerase, ThermoPrime™ Taq DNA polymerase, AccuPrime™ Taq DNA polymerase, Platinum™ Taq DNA polymerase, Platinum™ II Taq Hot-Start DNA polymerase, (Thermo Fisher Scientific™).

[0099] The DNA polymerases disclosed herein can also be derived from other organisms. As an alternative to isolation, some of the DNA polymerases disclosed herein are available commercially from, for example, Life Technologies (Carlsbad, CA), New England BioLabs (Beverly, MA), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, CA), Boehringer Mannheim Biochemicals (Indianapolis, IN) and Perkin Elmer Cetus (Norwalk CT). It is to be understood that a variety of DNA polymerases known to those of ordinary skill in the art can be used in the present PCR mixtures, including polymerases not specifically disclosed herein.

[0100] In exemplary embodiments, the concentration of a thermostable DNA polymerase in the PCR mixtures disclosed herein is about 0.01 to about 500 units per microliter, about 0.01 to about 50 units per microliter, about 0.01 to about 25 units per microliter, about 0.01 to about 10 units per microliter, about 0.1 to about 5 units per microliter, about 0.1 to about 2 units per microliter, about 0.1 to about 1 unit per microliter or about 0.1 to about 0.5 units per microliter (units per microliter = U / pL), including all concentrations and concentration ranges within any of the forgoing.

[0101] In exemplary embodiments, the PCR mixture includes a potassium salt, a magnesium salt, and / or a sodium salt. In exemplary embodiments, the salt concentration of the PCR mixture is about 5 mM to about 200 mM.

[0102] In exemplary embodiments, the PCR mixtures disclosed herein include an antifoam agent. An antifoam agent, as used herein, is a surface acting chemical which acts to facilitate gas release and to counteract foaming caused, for example, by mixing a reaction. Antifoam agents can prevent, eliminate, and / or reduce foam. Antifoam agents can additionally impart positive ancillary surface properties, such as wetting, dispersion, emulsification, and solubilization. In exemplary embodiments, the antifoam agent in the PCR mixtures is not a detergent. In exemplary embodiments, a combination of different antifoam agents is present inthe PCR mixtures described herein. In exemplary embodiments, the antifoam agent or agents is / are present in the PCR mixtures in an amount effective to enhance sensitivity of a PCR reaction.

[0103] Many chemical compounds can be used as antifoam agents, including without limitation, alkyl polyoxyalkylene glycol ethers, esters, alcohols, siloxanes, silicons, sulfites, sulfonates, fatty acids and their derivatives. A variety of such agents are known and commercially available from sources including J. T. Baker, Spectrum Chemicals, Dow Corning Corporation, and Sigma-Aldrich Company. Antifoam compositions can include a single component or multiple components which can be combined by simply mixing together. In exemplary embodiments, antifoam agents for use in the provided PCR mixtures include, without limitation, siloxane polymers, mixtures of organic non-silicone polypropylene based polyether dispersions, silicone emulsions, non-ionic simethicone emulsions, organic fatty acid ester-type antifoams. For example, organosiloxanes, a well-known class of silicone-based antifoam agents, include pure silicon oils such as dimethylpolysiloxanes as well as polysiloxane / polyoxyalkylene block copolymers such as dimethylpolysiloxanes. In exemplary embodiments, the antifoam agent is a silicon-based antifoam agent and is selected from XIAMETER™ AFE-1010, AFE- 1430, AFE-1510, AFE-1520, AFE-2210, Antifoam A, Antifoam B, Antifoam C, Antifoam H-10, Antifoam SE-15, Antifoam SE-35, Antifoam SO-25, Antifoam Y-30, and Antifoam 289.

[0104] In exemplary embodiments, the PCR mixture includes a silicone-based antifoam agent. In exemplary embodiments, the silicone-based antifoam agent is selected from XIAMETER™ AFE-1010, AFE-1430, AFE-1510, AFE-1520, AFE-2210, Antifoam A, Antifoam B, Antifoam C, Antifoam H-10, Antifoam SE-15, Antifoam SE-35, Antifoam SO-25, Antifoam Y-30, and Antifoam 289.

[0105] In exemplary embodiments, the PCR mixture includes a non-silicone-based antifoam agent. In exemplary embodiments, the non-silicone-based antifoam agent is selected from organic sulfonates, poly ethers, organic phosphates, acetylenic glycols, fluorocarbons, polyalkene polyamines, and polyalkyleneimine compounds, including without limitation Antifoam 204 and Antifoam 0-30.

[0106] In exemplary embodiments, the concentration of the antifoam agent in the PCR mixture is about 0.0005% to about 0.5%, about 0.001% to about 0.1%, 0.002% to about 0.05%, or 0.005% to about 0.02%, or about 0.01% to about 0.05%, including all concentrations and concentration ranges within any of the forgoing.

[0107] In exemplary embodiments, the PCR mixtures disclosed herein include at least one deoxynucleotide triphosphates (dNTP). In exemplary embodiments, the PCR mixture includes a combination of one or more deoxyribonucleotide triphosphates (dNTPs) and one or more dNTP derivatives. In exemplary embodiments, the PCR mixture includes two to eight different dNTPs and / or dNTP derivatives. In exemplary embodiments, the PCR mixture includes two, three, four, five, or six different dNTPs and / or dNTP derivatives. Examples of dNTPs which can be included in the PCR mixtures disclosed herein include, but are not limited to, dATP, dCTP, dGTP, dTTP, dUTP, and / or diTP. Examples of possible dNTP derivatives include, but are not limited to, 7-deaza-dGTP (such as 7-deaza-2-deoxy-dGTP), 7-deaza-dATP, alpha-thio-dATP, alpha-thio-dTTP, alpha-thio-dGTP, and / or alpha-thio-dCTP. In exemplary embodiments, the PCR mixture includes a combination of one or more deoxyribonucleotide triphosphates (dNTPs) and one or more dNTP derivatives. In exemplary embodiments, the PCR mixture includes one or more dideoxyribonucleotide triphosphates (ddNTPs) and / or one or more ddNTP derivatives. dNTPs, ddNTPs, and derivatives of each thereof, are available commercially from sources including Thermo Fisher Scientific, New England Biolabs, and Sigma-Aldrich Company. Such dNTPs, ddNTPs, and derivatives of each thereof may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g., 3H, 14C, 32P or 35S), vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin (DIG) and the like. Labeled dNTPs, ddNTPs, and derivatives of each thereof may also be obtained commercially, for example from Life Technologies (Carlsbad, CA) or Sigma Chemical Company (Saint Louis, MO).

[0108] The concentration of individual dNTPs, ddNTP, and / or derivatives of each thereof in the PCR mixtures disclosed herein do not need to be identical. In exemplary embodiments, dNTPs and / or ddNTPs can be added to the PCR mixture to give a concentration of each dNTP and / or ddNTP of about .001 mM to about 100 mM, about 0.01 mM to about 10 mM, about 0.1mM to about 1 mM, or preferably about 0.2 mM to about 0.8 mM, including any concentrations or range of concentrations within any of the forgoing. In exemplary embodiments, concentrations of each dNTP and / or ddNTP in the PCR mixture is such so that its final concentration during the rapid PCR is about 0.015 mM to about 5 mM, about 0.05 to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.15 mM to about 0.65 mM, about 0.15 mM to about 0.35 mM, or about 0.35 mM to about 0.65 mM, including any concentrations or range of concentrations within any of the forgoing. In exemplary embodiments, the PCR mixture includes dNTP derivatives and / or ddNTP derivatives, wherein the concentration of each dNTP derivative and / or ddNTP derivative of about .001 mM to about 100 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, or preferably about 0.2 mM to about 0.8 mM, including any concentrations or range of concentrations within any of the forgoing. In exemplary embodiments, concentrations of each dNTP and / or ddNTP in the PCR mixture is such so that its final concentration during the rapid PCR is about 0.015 mM to about 5 mM, about 0.05 to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.15 mM to about 0.65 mM, about 0.15 mM to about 0.35 mM, or about 0.35 mM to about 0.65 mM, including any concentrations or range of concentrations within any of the forgoing.

[0109] In exemplary embodiments, the PCR mixture includes a combination of a dNTP and a corresponding derivative of the same nucleotide thereof, such as a combination of a dNTP and the corresponding alpha-thio-dNTP derivative of the same nucleotide or a combination of a dNTP and the corresponding deaza-dNTP derivative of the same nucleotide. For example, in exemplary embodiments, the PCR mixture includes both dGTP and alpha-thio-dGTP. In exemplary embodiments, the PCR mixture includes both dGTP and 7-deaza-dGTP. In exemplary embodiments, the PCR mixture includes a combination of dATP and 7-deaza-dATP.

[0110] In exemplary embodiments, when both a dNTP and a derivative thereof are present in the PCR mixture, the relative concentration or concentration ratio of dNTP to derivative can vary. For example, in exemplary embodiments, the relative concentration of dNTP:dNTP derivative is 1 : 1. In exemplary embodiments, the dNTP concentration: dNTP derivative concentration is from about 100: 1 to about 1: 1, from about 50: 1 to about 1.2: 1, from about 25: 1 to about 1.5: 1, from about 10: 1 to about 2: 1. In exemplary embodiments, the dNTP concentration: dNTP derivative concentration is from about 1 : 1 to about 1: 100, from about 1 : 1.2to about 1 :50, from about 1 :25 to about 1 :1.5, from about 1 : 110 to about 1 :2. In exemplary embodiments, the dNTP concentration:dNTP derivative concentration is from about 2: 1 to about 1 :2. In exemplary embodiments, the dNTP concentration: dNTP derivative concentration is from about 10: 1 to about 2: 1. In exemplary embodiments, the dNTP concentration: dNTP derivative concentration is from about 1:2 to about 1 : 10. For example, in exemplary embodiments, the dGTP concentration :alpha-thio-dGTP concentration is from about 100: 1 to about 1.5: 1 or from about 50: 1 to about 12: 1. In exemplary embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 1: 1.5 to about 1 : 100 or from about 1 : 12 to about 1 :50. In exemplary embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 2: 1 to about 1 :2. In exemplary embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 10: 1 to about 2: 1. In exemplary embodiments, the dGTP concentration:alpha-thio-dGTP concentration is from about 1 :2 to about 1 : 10. In exemplary embodiments, the dGTP concentration:7-deaza-dGTP concentration is from about 100: 1 to about 1.5: 1 or from about 50: 1 to about 12: 1. In exemplary embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 1 : 1.5 to about 1 : 100 or from about 1 : 12 to about 1 :50. In exemplary embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 2: 1 to about 1:2. In exemplary embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 10: 1 to about 2: 1. In exemplary embodiments, the dGTP concentration: 7-deaza-dGTP concentration is from about 1 :2 to about 1 : 10.

[0111] In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a magnesium salt, an antifoam agent, and a combination of dATP, dCTP, dUTP, and 7-deaza-dGTP. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a magnesium salt, an antifoam agent, and a combination of dCTP, dGTP, dUTP, and 7-deaza-dATP. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a potassium salt, an antifoam agent, and a combination of dGTP and 7-deaza-dGTP. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a potassium salt, an antifoam agent, and a combination of dATP and 7-deaza-dATP. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a magnesium salt, an antifoam agent, and a combination of dGTP and 7-deaza-dGTP. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a magnesium salt, an antifoam agent, and a combination of dATP and 7-deaza-dATP. In exemplary embodiments, the antifoam agentis a silicone-based antifoam agent. Tn exemplary embodiments, the silicone-based antifoam agent is selected from XIAMETER™ AFE-1010, AFE-1430, AFE-1510, AFE-1520, AFE-2210, Antifoam A, Antifoam B, Antifoam C, Antifoam H-10, Antifoam SE-15, Antifoam SE-35, Antifoam SO-25, Antifoam Y-30, and Antifoam 289. In exemplary embodiments, the antifoam agent is a non-silicone-based antifoam agent. In exemplary embodiments, the non-silicone-based antifoam agent is selected from organic sulfonates, polyethers, organic phosphates, acetylenic glycols, fluorocarbons, polyalkene polyamines, and polyalkyleneimine compounds, including without limitation Antifoam 204 and Antifoam 0-30.

[0112] In exemplary embodiments, the PCR mixture includes at least one or more dNTPs selected from dGTP, dCTP, dATP and / or dTTP. In exemplary embodiments the PCR mixture includes dGTP. In exemplary embodiments, the PCR mixture contains a dNTP concentration of 0.05 mM to 1.0 mM.

[0113] In exemplary embodiments, the PCR mixture includes a dNTP derivative selected from 7-deaza-2-deoxy-GTP, 7-deaza-dATP, alpha-thio-dATP, alpha-thio-dTTP, alpha-thio- dGTP, and alpha-thio-dCTP. In exemplary embodiments, the dNTP derivative is 7-deaza-2- deoxy-GTP. In other exemplary embodiments, the dNTP derivative is 7-deaza-2-deoxy-dGTP. In exemplary embodiments, the PCR mixture includes a dNTP derivative at a concentration of 0.05 mM to 1 .0 mM.

[0114] In exemplary embodiments, the PCR mixture includes a dGTP and a 7-deaza-2- deoxy-dGTP at a ratio of about 1 :2 to about 2: 1. In exemplary embodiments, the PCR mixture contains a dGTP and a 7-deaza-2-deoxy-dGTP at a ratio of about 1 :2 to about 1 : 10. In exemplary embodiments, the PCR mixture includes a dGTP and a 7-deaza-2-deoxy-dGTP at a ratio of about 2: 1 to about 10: 1. In exemplary embodiments, the PCR mixture includes a dGTP and a 7-deaza- 2-deoxy-dGTP at a ratio of about 1: 1.

[0115] In exemplary embodiments, the PCR mixture includes one or more PCR inhibitor blocking agents. In exemplary embodiments, the PCR inhibitor blocking agent is a protein. In exemplary embodiments, the PCR inhibitor blocking protein is selected from an albumin, a gelatin, and a combination thereof. In exemplary embodiments, the PCR mixture includes a gelatin and a serum albumin. In exemplary embodiments, the gelatin is selected from a bovinegelatin, a fish gelatin and a combination thereof. In exemplary embodiments, the PCR mixture includes a bovine gelatin at a concentration of 0.01% to 1.0% and / or a fish gelatin at a concentration of 0.01% to 1.0%. In exemplary embodiments, the serum albumin is a bovine serum albumin. In exemplary embodiments, the PCR mixture includes a bovine serum albumin at a concentration of 0.05 mg / ml to 5 mg / ml.

[0116] In exemplary embodiments, the PCR inhibitor blocking agent disclosed herein is a protein selected from, but not limited to, albumins, gelatins, DNA-binding proteins, peptide or polypeptide variants, fragments or derivatives thereof. In exemplary embodiments, other nonprotein-based PCR inhibitor blocking agents are included in the PCR mixture, for example, deferoxamine mesylate. In exemplary embodiments, the PCR mixtures include a combination of PCR inhibitor blocking agents and / or proteins. For example, in exemplary embodiments the PCR mixture includes an albumin, a gelatin, or a combination of albumin and gelatin. In exemplary embodiments, the albumin or gelatin is selected from serum albumin, fish gelatin, or a combination of serum albumin and fish gelatin. The serum albumin can be from any animal, e.g., bovine serum albumin (BSA), human serum albumin (HSA). In exemplary embodiments, the albumin is derived from other species of animals which are well-known to those of skill in the art. In exemplary embodiments, the albumin is a recombinant albumin, such as recombinant BSA (rBSA). The gelatin can be from any animal, e.g., fish gelatin, bovine gelatin. In exemplary embodiments, the gelatin is derived from other species of animals which are well- known to those of skill in the art. In exemplary embodiments, the gelatin is a recombinant gelatin, such as recombinant human gelatin. In exemplary embodiments, the DNA-binding proteins can include, but are not limited to T4 gene 32 protein (T4 gp32).

[0117] The PCR inhibitor blocking compounds or agents disclosed herein can be added to the PCR mixtures to give a concentration in the mixture of about .0001 mg / mL to about 10 mg / mL, about .001 mg / mL to about 8 mg / mL, about 0.01 mg / mL to about 6 mg / mL, about 0.05 mg / mL to about 4 mg / mL, about 0.1 mg / mL to about 3 mg / mL or about 0.5 mg / mL to about 2 mg / mL, including any concentrations or range of concentrations within any of the forgoing. The PCR inhibitor blocking agents disclosed herein can also be added as a percentage of the final concentration of the PCR mixture, for example, from about 0.001% to about 15%, about 0.05% to about 10%, about 0.01% to about 5%, or about 0.1% to about 1%, including anyconcentrations or range of concentrations within any of the forgoing. In exemplary embodiments, the PCR mixtures include a PCR inhibitor blocking protein, such as albumin, at a concentration such that its concentration during the rapid PCR is about 0.001 mg / mL to about 10.0 mg / mL, 0.005 mg / mL to about 5.0 mg / mL, about 0.01 mg / mL to about 4.0 mg / mL, about 0.05 mg / mL to about 3.0 mg / mL or about 0.1 mg / mL to about 2.0 mg / mL, including any concentrations or range of concentrations within any of the forgoing. In exemplary embodiments, the PCR mixtures disclosed herein include a PCR inhibitor blocking protein, such as gelatin, at a concentration such that its concentration during the rapid PCR is about 0.005% (w / v) to about 2% (w / v), about 0.01% (w / v) to about 1.0% (w / v), and more specifically about 0.05% (w / v) to about 0.5% (w / v), including any concentrations or range of concentrations within any of the forgoing. In exemplary embodiments, the PCR mixtures disclosed herein include a combination of albumin and gelatin at any of the foregoing concentrations. In exemplary embodiments, the albumin is at about 0.05 mg / mL to 5 mg / mL and gelatin is at a concentration of about 0.01% (w / v) to about 1%. In exemplary embodiments, the albumin is bovine serum albumin (BSA), and the gelatin is a fish gelatin and / or a bovine gelatin.[00118J In exemplary embodiments, the PCR mixtures include one, two, three, four, five or more different PCR inhibitor blocking proteins and / or agents. For example, in exemplary embodiments the PCR mixture includes an albumin, a fish gelatin and a bovine gelatin. In exemplary embodiments, the concentration of each PCR inhibitor blocking protein and / or agent is the same. In exemplary embodiments, the concentration of each PCR inhibitor blocking protein and / or agent is different. In exemplary embodiments, one or more PCR inhibitor blocking agents are added to the PCR mixture to help to overcome inhibition of PCR by a variety of inhibitors often found in biological samples, such inhibitors include, for example, heparin (blood); hematin (blood); EDTA (blood); citrate (blood); immunoglobin G (blood, serum); humic acid (soil, feces); lactoferrin (milk, saliva, other secretory fluids); urea (urine); plant polysaccharides (plants); melanin (skin, hair); myoglobin (tissue); and indigo dye (textiles). The addition of PCR inhibitor blocking agents, both individually and in combination, can increase tolerance to such PCR inhibitor contaminants. Thus, the PCR mixtures disclosed herein can also include agents that work alone or in combination to increase tolerance to various PCR inhibitors including, for example, humic acid, hematin, and heparin. In exemplary embodiments, each PCR inhibitor blocking agent or protein in the PCR mixture can block a different inhibitor or group ofinhibitors than the other PCR inhibitor blocking agent or protein in the PCR mixture. In exemplary embodiments, the different PCR inhibitor blocking agents or proteins in the PCR mixture can block the same inhibitor or group of inhibitors. In exemplary embodiments, the different PCR inhibitor blocking agents or proteins in the PCR mixture can block an overlapping number of inhibitor of a group of inhibitors. For example, in exemplary embodiments, gelatin is effective at reducing PCR inhibition by at least humic acid and heparin, and albumin is effective at reducing PCR inhibition by at least humic acid and hematin.

[0119] The PCR inhibitor blocking agents disclosed herein can be added to the PCR mixtures disclosed herein to assist in overcoming the inhibition of PCR reactions by a variety of compounds often found in biological samples. In exemplary embodiments, the PCR inhibitor blocking agent(s) can reduce the amount of PCR inhibition by a PCR inhibitor or inhibitors from some percentage above zero up to 100% compared to the level of inhibition observed in the absence of such PCR inhibitor blocking agents. For example, inhibition can be reduced by at least about 0.5%, about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 90%, about 95% or about 100% or any percentage in between.

[0120] In exemplary embodiments, the PCR mixtures disclosed herein include one or more primers which facilitate the synthesis of a DNA molecule (e.g., a single-stranded cDNA molecule or a double-stranded cDNA molecule) complementary to all or a portion of a nucleic acid template (RNA or DNA). Additionally, these primers can be used in amplifying nucleic acid molecules in accordance with the present disclosure. Oligonucleotide primers can be any oligonucleotide of two or more (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, and so on) nucleotides in length. Such primers include, but are not limited to, target-specific primers (which are preferably genespecific primers), oligo(dT) primers, random primers or arbitrary primers. Additional primers that can be used for amplification of the DNA molecules according to the methods disclosed herein will be apparent to one of ordinary skill in the art. It is to be understood that a vast array of primers known to those of skill in the art can be used in the present methods and PCR mixtures, including those not specifically disclosed herein.

[0121] In exemplary embodiments, the final concentration of primers in the PCR mixture can range from about 25 nM to about 2000 nM, such as about 50 nM to about 1700 nM, about 75nM to about 1500 nM, about 100 nM to about 1200 nM, about 200 nM to about 1000 nM, or any range in between. In exemplary embodiments, the concentration of each primer is between about 400 nM to about 900 nM, including all amounts or ranges in between.

[0122] In exemplary embodiments, the PCR mixture includes probes for the detection of target nucleic acids. Various probes are known in the art, for example (TaqMan™ probes (see, e.g., U.S. Pat. No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99 / 21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise™ / Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes (see, e.g., Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat.No. 6,589,743), bulge loop probes (see, e.g., U.S. Pat. No. 6,590,091), pseudo knot probes (see, e.g., U.S. Pat. No. 6,589,250), cyclicons (see, e.g., U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (see, e.g., U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik etal., 2000, Anal Biochem. 281 :26-35; Wolffs et al., 2001, Biotechniques 766:769-771 ;Tsourkas et al., 2002, Nucleic Acids Res. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Res. 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res. Toxicol. 15: 118-126; and Yu e / al., 2001, J. Am. Chem. Soc 14: 11155-11161. Probes can include reporter dyes such as, for example, 6-carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET). Detector probes can also include quencher moieties such as tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Thermo Fisher Scientific), and Dabsyl and Dabcel sulfonate / carboxylate Quenchers (Epoch). Probes can also include two probes, wherein for example a fluorophore is on one probe, and a quencher on the other, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on a target alters the signal signature via a change in fluorescence.

[0123] Exemplary detectable labels that can be included in the PCR mixtures include, but are not limited to, a fluorescent dye or fluorophore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like. Suitable detectable labels can include, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 6-HAT; 6-JOE; 6- carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY™ fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, FLCeramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxy coumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g., calcium crimson, calcium green, calcium orange, cal cofluor white), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., GFP. EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, BODIPY™ FL / BODIPYTM FL, Fluorescein / QSY7 and QSY9, QSY21), LysoTracker and LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue- White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND- 153, LysoSensor Yellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, 5-ROX (carboxy- X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC, JUN, ABY and other labels described in, e.g., US Publication No. 2009 / 0197254), among others as would be known to those of skill in the art.

[0124] In exemplary embodiments, the PCR mixtures includes detectable labels that (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label; or (iii) confers a capture function, e.g. hydrophobic affinity, antibody / antigen, ionic complexation. The skilled artisan will appreciate that many different species of reporter labels can be used in the present disclosure, either individually or in combination with one or more different labels. Exemplary labels include, but are not limited to, fluorophores, radioisotopes, quantum dots, chromogens, enzymes, antigens including but not limited to epitope tags, heavy metals, dyes, phosphorescence groups, chemiluminescent groups, electrochemical detection moieties, affinity tags, binding proteins, phosphors, rare earth chelates, and near-infrared dyes.

[0125] In exemplary embodiments, the probes are designed according to the methods and principles described in, for example, U.S. Patent No. 6,727,356 (the disclosure of which is incorporated herein by reference in its entirety). Some probes can be sequence-based, for example, 5' nuclease probes and some, such as SYBR™ Green can be non-sequence specific DNA-binding dyes. In exemplary embodiments, the detector probe is a TaqMan™ probe (Applied Biosystems, Foster City, CA). It is to be understood that a wide variety of probes are known in the art that can be used in the present methods and PCR mixtures, including those not specifically disclosed herein.

[0126] In exemplary embodiments, the probe concentration in the PCR mixture can range from about 5 nM to about 750 nM, such as about 10 nM to about 600 nM, about 25 nM to about 500 nM, about 50 nM to about 400 nM, about 75 nM to about 300 nM, or any number in between. In exemplary embodiments, the concentration of each probe in the PCR mixture is between about 100 nM to about 250 nM, including all amounts or ranges in between.

[0127] In exemplary embodiments, the PCR mixture includes fluorescent reporter molecule— quencher molecule pairs incorporated onto oligonucleotide probes. These molecule pairs can detect biological events through the movement of the fluorescent reporter molecule and quencher molecule. For example, probes have been developed where the intensity of the reporter molecule fluorescence increases due to the separation of the reporter molecule from the quencher molecule. Probes have also been developed which lose their fluorescence because the quenchermolecule is brought into proximity with the reporter molecule. These reporter-quencher molecule pair probes can be used to monitor hybridization assays and nucleic acid amplification reactions, especially polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the fluorescence signal generated by the reporter molecule, (see, e.g., U.S. Pat. No. 6,030,787). Exemplary reporter-quencher pairs can be selected from xanthene dyes, including fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are widely available commercially with substituents on their phenyl moieties, which can be used as the site for bonding or as the bonding functionality for attachment to an oligonucleotide. Another group of fluorescent compounds that can be used are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1- dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6- naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines, such as 9- isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl) maleimide; benzoxadiazoles, stilbenes, and pyrenes. In exemplary embodiments, the reporter and quencher molecules are selected from fluorescein and rhodamine dyes. These dyes and appropriate linking methodologies for attachment to oligonucleotides are described in many references, e.g., Marshall, Histochemical J., 7: 299-303 (1975); Menchen et al., U.S. Pat. No. 5,188,934;Menchen et al., European Patent Application 87310256.0; and Bergot et al., International Application PCT / US90 / 05565.

[0128] In exemplary embodiments, the PCR mixture includes one or more additional components and / or additives capable of facilitating or enhancing nucleic acid synthesis reactions (e.g., reagents for facilitating or enhancing PCR). Components and / or additives which enhance nucleic acid synthesis can be organic or inorganic compounds. Some components and / or additives that enhance nucleic acid synthesis include polypeptides, as well as nonpolypeptide components. Such components and / or additives can include, but are not limited to, singlestranded binding DNA-binding (SSB) proteins, sulfur-containing compounds, acetate-containing compounds, dimethylsulfoxide (DMSO), glycerol, formamide, betaine, tetramethylammonium chloride (TMAC), ectoine, sodium azide, kathon, polyols, NaNs, buffers, surfactants, detergents (e.g., TWEEN 20, Tritin X-100, and CHAPS), a component or compound used for hot start PCR, a passive reference control to minimize sample-to-sample and / or well-to-well variations in quantitative real-time DNA-detection assays, and / or uracil DNA glycosylase. In exemplaryembodiments, the PCR mixture includes crowding agents, such as Ficoll 70, glycogen, and polyethylene glycol (PEG). Those of ordinary skill in the art will be able to identify additional reagents and / or additives capable of facilitating or enhancing nucleic acid synthesis reactions, including those not specifically disclosed herein.

[0129] In exemplary embodiments, the PCR mixture includes a detergent or a combination of detergents. In exemplary embodiments, the detergent is a non-ionic detergent. In exemplary embodiments, the non-ionic detergent is selected from the group consisting of TRITON X-100™, TWEEN 20, TWEEN 80, Brij 30, Brij 35, and Brij 58. In exemplary embodiments, the non-ionic detergent is a detergent other than Tween-20. In exemplary embodiments, the non-ionic detergent is present in the PCR mixture at a concentration of .005% to 0.1%. In exemplary embodiments, the detergent(s) is / are present in an amount effective to enhance sensitivity of a PCR reaction. The detergent(s) can be an anionic detergent, a cationic detergent, a zwitterionic detergent, and / or a nonionic detergent. In exemplary embodiments, the PCR mixture includes TRITON X-100™. In exemplary embodiments, the PCR mixture includes Brij 30, Brij 35, or Brij 58. In exemplary embodiments, the PCR mixture includes any combination of the aforementioned detergents. The detergent(s) can be present in the PCR mixture at a concentration that is about 0.001% to about 0.50% (w / v), about 0.005 to about 0.1% (w / v), or about 0.01% (w / v) to about 0.05% (w / v), including all concentrations or range of concentrations in between any of the foregoing amounts.

[0130] In exemplary embodiments, the PCR mixture includes a buffer component, such as a buffered salt solution. In exemplary embodiments, the buffer agent provides appropriate pH conditions to maintain stability of a DNA polymerase enzyme in the PCR mixture. The terms "stable" and "stability" as used herein generally mean the retention by a composition, such as an enzyme composition, of at least 70%, preferably at least 80%, and most preferably at least 90%, of the original enzymatic activity (in units) after the enzyme or composition containing the enzyme has been stored for about 3 days at a temperature of about room temperature (e.g, about 20°C to about 25°C), about one to eight weeks at a temperature of about 4°C, about two to six months at a temperature of about -20°C, and about six months or longer at a temperature of about -80 °C. Examples of such buffering agents can include, but are not limited to, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES TAPS, PIPES, and CAPS. In exemplary embodiments, thebuffer concentration in the PCR mixtures disclosed herein is between about 1 mM and about 500 mM, between about 5 mM and about 250 mM, between about 10 mM and about 200 mM, between about 25 mM and about 150 mM, and between about 50 mM and about 100 mM, including any concentration or range falling within the forgoing amounts. It is to be understood that a wide variety of buffers (or buffer salts) are known in the art can be used in accordance with the present methods and PCR mixtures, including those not specifically disclosed herein.

[0131] In exemplary embodiments, the PCR mixture includes one or more salts. Examples of salts suitable for inclusion in the PCR mixtures disclosed herein include, without limitation, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride and lithium acetate. In exemplary embodiments, the PCR mixture includes more than one (e.g., two, three, four, five, six, etc.) salt component. For example, the PCR mixtures described herein can include two different salts. In exemplary embodiments, the PCR mixture includes three different salts. In exemplary embodiments, the PCR mixture includes four different salts. In exemplary embodiments, the PCR mixture includes a particular salt at a concentration of about 0.5 mM to about 1000 mM, about 1 mM to about 500 mM, about 5 mM to about 250 mM, about 10 mM to about 100 mM, about 5 mM to about 50 mM, and about 2 mM to about 10 mM, including any concentration or range falling within the forgoing ranges. Without limitation, in exemplary embodiments where the PCR mixture includes a salt such as potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and / or ammonium acetate, the PCR mixture also includes the salt at a concentration, such that during the rapid PCR, the salt concentration is about 5 mM to about 250 mM, 5 mM to about 150 mM, about 10 mM to about 120 mM, about 20 mM to about 100 mM, about 30 mM to about 90 mM, or about 40 to about 80 mM, including any concentration falling within the forgoing ranges. Without limitation, in exemplary embodiments where the PCR mixture includes a salt such as magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, or manganese sulfate, the PCR mixture includes the salt at a concentration, such that during the rapid PCR, the salt concentration is about 0.5 mM to about 100 mM, about 1 mM to about 75 mM, about 1.5 mM to about 50 mM, about 2 mM to about 30 mM, about 3 mM to about 15 mM, about 4 mM to about 10 mM, or about 1 mM to about 5 mM, including anyconcentration falling within the forgoing ranges. It is to be understood that a wide variety of salts or salt solutions are known in the art that can be used in accordance with the present methods and PCR mixtures, including those not specifically disclosed herein.

[0132] In exemplary embodiments, the PCR mixture includes glycerol. In exemplary embodiments, glycerol is present in the PCR mixture at a concentration of about 5 to about 50% (w / v), or from about 10% (w / v) to about 30% (w / v), including all concentrations or range of concentrations in between any of the foregoing amounts.

[0133] In exemplary embodiments, the PCR mixture includes NaNa. In exemplary embodiments, NaNa is present in the PCR mixture at a concentration of about 0.005% (w / v) to about 0.05% (w / v), or from about 0.01% (w / v) to about 0.1% (w / v).

[0134] In exemplary embodiments, the PCR mixture includes a passive reference control component. In exemplary embodiments, the passive reference control component is a passive reference control dye. In exemplary embodiments, the passive reference control dye is a fluorescent dye. In exemplary embodiments, the fluorescent passive reference dye is a ROX™ dye or a MUSTANG PURPLE™ dye. In exemplary embodiments, the PCR mixture contains a passive reference dye at a concentration of about 25 nM to about 500 nM. In exemplary embodiments, the passive reference control minimizes sample-to-sample and / or well-to-well variations in quantitative real-time nucleic acid-detection assays and / or can be included at a concentration allowing its use as detectable control. In exemplary embodiments, a reference chromophore, specifically a fluorophore, is included as the passive reference control. In exemplary embodiments, the reference chromophore is a fluorescent dye. In exemplary embodiments, the fluorescent dye is a ROX™ dye (Thermo Fisher Scientific). In exemplary embodiments, the fluorescent dye is a MUSTANG PURPLE™ dye (Thermo Fisher Scientific). The passive reference control component can be included in the PCR mixture at a concentration, such that its concentration during the rapid PCR, is about 10 nM to about 750 nM, or about 20 nM to about 500 nM, or about 50 nM to about 200 nM, including any concentration falling within the forgoing ranges.

[0135] In exemplary embodiments, uracil DNA glycosylase (UNG or UDG) is included in the PCR mixtures disclosed herein. This enzyme is commercially available from a number ofcommercial sources, for example Thermo Fisher Scientific, Enzymatics, New England Biolabs, Genscript, or USB. In exemplary embodiments, UNG is thermolabile. In exemplary embodiments, UNG is thermostable. Thermolabile or thermostable UNG can be included in the PCR mixture at a concentration, such that its concentration during the rapid PCR, is about 0.0001 U / pL to about 50 U / pL, about 0.0005 U / pL to about 10 U / pL, about 0.001 U / pL to about 5 U / pL, 0.005 U / pL to about 1.0 U / pL, or 0.01 U / pL to about 0.5 U / pL (U / pL = units per microliter), including any concentration or range falling within the forgoing ranges.

[0136] In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, potassium chloride, an antifoam agent containing silicon, dGTP and 7-deaza-2- deoxy-dGTP, where the 7-deaza-2-deoxy-dGTP is at a concentration of about 0.05 mM to 1.0 mM, a non-ionic detergent other than Tween-20, bovine gelatin at a concentration of 0.01% to 1.0%, fish gelatin at a concentration of 0.01% to 1.0%, and bovine serum albumin at a concentration of 0.05 mg / ml to 5.0 mg / ml.

[0137] In exemplary embodiments, the PCR mixture includes “hot start” components as a means to further prevent, reduce or eliminate nonspecific nucleic acid synthesis. In exemplary embodiments, the PCR mixture includes components or compounds used for hot start reactions that can be any of those which prevent non-specific amplification of DNA by inactivating polymerase activity at lower temperature, such as during the annealing phase, while allowing reactivation or activation of the polymerase activity at a higher temperature, such as during the extension phase. Such examples of hot start components can include, but are not limited to, for example, an antibody, a chemical modification (e.g., of the polymerase), an oligonucleotide, an aptamer, a specially-designed primer, a binding protein, and / or a sequestration wax bead. Wax beads for hot start PCR are commercially available, e.g., HotStart ™ Storage reaction tubes (Thermo Fisher Scientific). Selection of a suitable hot start aptamer can be performed by a method known in the art or a commercially available hot start aptamer can be used. Similarly, selection of a suitable hot start hairpin primer can be performed by a method known in the art or a commercially available hot start primer can be used. Antibodies for hot start PCR can be generated or selected by various methods known in the art. Alternatively, a commercially available antibody can be used, for example, the TaqStart Antibody (Clontech) which is effective with any Taq-derived DNA polymerase, including native, recombinant, and N-terminal deletionmutants. In exemplary embodiments, the PCR mixture includes a suitable hot start primer that has been specially designed to have a secondary structure (such as a hairpin primer) which prevents the primer from annealing until cycling temperatures cause them to denature and unfold. An appropriate concentration of the compound or reagent for hot start PCR in the PCR mixtures can be determined by a number of methods known in the art or, for a commercial product, suggested by the manufacturer.

[0138] In exemplary embodiments, the PCR mixtures disclosed herein include a thermostable DNA polymerase, a salt, an antifoam agent, dNTPs, a combination of PCR inhibitor blocking agents, a buffer, a hot start component, glycerol, a nonionic detergent, and a passive reference dye. In exemplary embodiments, the PCR mixture includes a thermostable DNA polymerase, a potassium salt, a magnesium salt, a silicon-based antifoam agent, dNTPs including a dNTP / dNTP derivative combination, an albumin, fish gelatin, a buffer, a hot start component, glycerol, a nonionic detergent other than TWEEN 20, and a passive reference dye. The components can be substituted or modified as determined by those of skill in the art. It is to be understood that a wide variety of additional components known in the art can be useful in the present methods and PCR mixtures, including those not specifically disclosed herein. Those of skill in the art will also understand the methods required to determine the particular conditions or concentrations for optimal use of each component in the PCR mixture.

[0139] In exemplary embodiments, the PCR mixtures are provided as a concentrated stock solution or mix. As used herein, the term "concentrated stock" means at a concentration that requires further dilution in order to achieve optimal concentration for use in a solution to perform a particular function (such as PCR amplification). For example, the PCR mixture can be stock solutions of about 2X, about 3X, about 4X, about 5X, about 6X, about 10X, and so on, including any amount between any of the forgoing. In exemplary embodiments, the PCR mixture can require greater than 2X, greater than 3X, greater than 4X, greater than 5X, greater than 6X, greater than 10X, and so on, including any amount between any of the forgoing, dilution to be at working, or optimal, concentration for use, for example, in nucleic acid synthesis or amplification methods. In exemplary embodiments, the PCR mixtures disclosed herein can also be provided at a working concentration or as a “working solution or mix.” As used herein “working concentration” or “working solution or mix” is used to refer to a solutionor mix that is at or near the optimal concentration used to perform a particular function or reaction (such as amplification or PCR). In exemplary embodiments, the PCR mixture is at a working concentration that needs no or minimal dilution prior to use. For example, in exemplary embodiments, the PCR mixture is at a working concentration of about IX, about 1.25X, about 1.5X, about 1.8X, about 2X, or about 2.2X.

[0140] In exemplary embodiments, the PCR mixtures are formulated as master mixes. Master mixes can improve efficiency and reduce errors associated with the assembly of a large number of reactions required for high-throughput analysis. In exemplary embodiments, master mixes can contain a combination of reagents common to all reactions. For example, in exemplary embodiments, the master mix contains a buffer, a salt, such as MgCh, deoxynucleotide triphosphates (dNTPs), a thermostable DNA polymerase, a detergent, and a PCR inhibitor blocking agent. For use in certain assays, each reaction would then contain an aliquot of the common master mix and a specific target nucleic acid template and at least one primer. In exemplary embodiments, the PCR mixture master mixes are manufactured and distributed as a concentrated stock solution or mixture. The PCR mixture master mixes can then be diluted when final reaction mixtures are assembled. In exemplary embodiments, the PCR mixture master mix is manufactured and distributed as a working solution or mixture. In embodiments, the master mix is a master mix formulated for higher multiplexing (e.g., TaqPath™ ProAmp™ Multiplex master mix by Thermo Fisher Scientific™).

[0141] In exemplary embodiments, the PCR mixtures disclosed herein are packaged in a suitable container capable of holding the PCR mixture and which will not significantly interact with components of the mixture. The container can be one designed to permit easy dispensing of the dosage form by individuals or by a liquid handling instrument. The containers of the PCR mixtures can also be packaged into multi-pack units. In exemplary embodiments, the multi-pack units contain additional containers including additives or other reagents to be added to the PCR mixture prior to use in the rapid PCR.

[0142] In exemplary embodiments, the PCR mixtures disclosed herein are in a liquid form, such as in a hydrated solution. In exemplary embodiments, the PCR mixtures disclosed herein are in a gel form. A “gel” as used herein is a composition which is not solid or frozen at -20°C. In exemplary embodiments, the PCR mixtures disclosed herein are in a dehydrated or dried form, such as in a lyophilized composition.

[0143] In exemplary embodiments, the PCR mixtures disclosed herein are used in simplex PCR procedures. In exemplary embodiments, the PCR mixtures disclosed herein are used in multiplex PCR procedures.

[0144] In exemplary embodiments, the PCR mixture includes deoxynucleotide triphosphates, magnesium ions, potassium ions, a buffer solution, at least one primer pair specific for nucleotides in a target nucleic acid, at least one probe specific for nucleotides in the target nucleic acid, and at least one thermostable polymerase, wherein the at least one probe includes a detectable label. In exemplary embodiments, the target nucleic acid is a biomarker for a disease.

[0145] In exemplary embodiments, the PCR mixture includes and at least one thermostable polymerase, wherein the at least one thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, Platinum Taq DNA polymerase, Platinum II Taq Hot-Start DNA polymerase, AmpliTaq DNA polymerase, Invitrogen Taq DNA, Dream Taq DNA polymerase and mutants or derivatives thereof having DNA polymerase activity, as well as those discussed above.

[0146] The PCR mixtures disclosed herein can provide superior performance sensitivity, accuracy, and specificity when compared with standard PCR reagent mixes. In exemplary embodiments, use of the PCR mixtures disclosed herein with the crude nucleic acid sample result in equivalent or better amplification, detection, and / or quantitation of a nucleic acid target molecule present in the crude nucleic acid sample as compared with use of standard PCR compositions with a processed nucleic acid sample (e.g., a diluted and / or lysed nucleic acid sample). In exemplary embodiments, use of the PCR mixtures disclosed herein result in equivalent or better amplification, detection, and / or quantitation of a nucleic acid target molecule present in low copy number in the crude nucleic acid sample as compared with use of standardPCR master mix compositions with a processed nucleic acid sample (e.g., a diluted and / or lysed nucleic acid sample).

[0147] In exemplary embodiments, the crude nucleic acid sample is incubated in the PCR mixture for a set period of time. For instance, the crude nucleic acid sample may be incubated in the PCR mixture at 60°C for about 10 seconds.

[0148] In exemplary embodiments, the performing rapid PCR further includes performing a pre-PCR read of the incubated crude nucleic acid sample, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle.

[0149] In exemplary embodiments, the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

[0150] In exemplary embodiments, the first plurality of amplification cycles includes four amplification cycles where each cycle includes performing the second denaturation for about one second followed by annealing and extension for 3 seconds. In exemplary embodiments, the at least one second amplification cycle includes performing the second denaturation for about one second followed by annealing and extension for 10 seconds.

[0151] In exemplary embodiments, the performing rapid PCR on the crude nucleic acid sample includes subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle 8 times.

[0152] In exemplary embodiments, a thermal cycling and data collection method are used to enable a shortened instrument run time, by conducting a plurality of amplification cycles without data collection followed by at least one amplification cycle with data collection. Theshortened run time is accomplished by subsampling data collection at, for example, every fifth rapid PCR cycle. For instance, data collection is performed on the 5thcycle, the 10th cycle, and so forward, while no data is collected on the lst-4thcycles and 6th-9thcycles, and so forward. However, example embodiments are not limited thereto. That is, the number of amplification cycles without data collection and / or the at least one amplification cycle with data collection can be adjusted depending on desired rapid PCR run time.

[0153] In exemplary embodiments, a thermal cycling and data collection method are used to enable a shortened instrument run time, by conducting a plurality of amplification cycles with a shorter denaturation time (1-2 seconds, or preferably 1 second) and / or a shorter annealing and extension time (2-5 seconds, or preferably, 3 seconds) and without data collection, followed by at least one amplification cycle with the shorter denaturation time (1-2 seconds, or preferably 1 second) and / or a longer annealing and extension time (e.g., 8-11 seconds, or preferably 10 seconds) with data collection. However, example embodiments are not limited thereto. That is, the denaturation, annealing and extension times of the amplification cycles without data collection and / or the at least one amplification cycle with data collection can be adjusted depending on desired rapid PCR run time.

[0154] In exemplary embodiments, the run time of the rapid PCR performed using the PCR mixtures and parameters as described herein is decreased compared to an equivalent PCR involving standard PCR reaction mixes and sample preparation. In exemplary embodiments, the rapid PCR performed using the PCR mixtures and parameters as described herein produces an amplification product in a shorter period of time compared to an equivalent PCR involving a standard PCR reaction mix and sample preparation.

[0155] In exemplary embodiments, the rapid PCR is performed in less than 30 minutes. In exemplary embodiments, the rapid PCR is performed in about 23-27 minutes.

[0156] In exemplary embodiments, the rapid PCR can be used to detect, without limitation, a single nucleotide polymorphism (SNP), a microsatellite, copy number variation, gene expression, and / or small RNA expression (e.g., microRNA expression).

[0157] In exemplary embodiments, the rapid PCR employes a hydrolysis probe for the detection of nucleic acids. Hydrolysis probes take advantage of the 5' exonuclease activity of some polymerases. During the extension or elongation phase of a PCR reaction, a polymerase, such as Taq polymerase, uses an upstream primer as a binding site and then extends. The hydrolysis probe is then cleaved during polymerase extension at its 5' end by the 5'-exonuclease activity of the polymerase.

[0158] In exemplary embodiments, the rapid PCR includes performing a TaqMan™ assay (see, e.g., U.S. Patent 5,210,015, incorporated herein by reference in its entirety). A TaqMan™ assay is an example of a hydrolysis-probe based assay. In the TaqMan™ assay, hydrolysis probes are labeled with a reporter on the 5' end and a quencher on the 3' end. When the reporter and quencher are fixed onto the same probe, they are forced to remain in close proximity. This proximity effectively quenches the reporter signal, even when the probe is hybridized to the target sequence. The hydrolysis probes are cleaved during polymerase extension at their 5' end by the 5'-exonuclease activity of Taq. When this occurs, the reporter fluorophore is released from the probe, and subsequently, is no longer in close proximity to the quencher. This produces a perpetual increase in reporter signal with each extension phase as the PCR reaction continues cycling. To achieve maximal signal with each cycle, hydrolysis probes are often designed with a Tm that is roughly 10°C higher than the primers in the reaction. Uses of the real-time hydrolysis probe reaction are also described in U.S. Patent Nos. 5,538,848, 6,653,473, 7,485,442 and 7,205,105, the disclosures of all of which are incorporated herein by reference in their entireties. In exemplary embodiments, the rapid PCR includes performing at least one or more of the following assays: gene expression assays (e.g., TaqMan™ Gene Expression Assays), copy number variation assays (e.g., TaqMan™ Copy Number Assays), genotyping assays (e.g., TaqMan™ Drug Metabolism Genotyping Assays or TaqMan™ SNP Genotyping Assays), miRNA assays (e.g., TaqMan™ MicroRNA Assays) or RNA quantitation assays (e.g., two-step reverse transcription-polymerase chain reaction assays), and TaqMan™ Low Density Array Assays.

[0159] In exemplary embodiments, the rapid PCR is performed in a single rection vessel. As used herein, the term “reaction vessel” generally refers to any container in which a reaction can occur in accordance with the disclosed methods. In exemplary embodiments, the reactionvessel is a microtube, for example, but not limited to, a 0.2 mb or a 0.5 mL reaction tube such as a MicroAmp™ Optical tube (Applied Biosystems™, Thermo Fisher Scientific™) or a microcentrifuge tube, or other containers of the sort in common practice in molecular biology laboratories. In exemplary embodiments, the reaction vessel is a well in a microtiter plate (e.g., 96-well plate, 384-well plate) such as a TaqMan™ Array plate (Applied Biosystems™; Thermo Fisher Scientific™), a spot on a glass slide, a well in an Applied Biosystems™ TaqMan™ Array Card or Plate (Thermo Fisher Scientific™) or a through-hole of an Applied Biosystems™ TaqMan™ OpenArray™ plate (Thermo Fisher Scientific™). For example, a plurality of reaction vessels can reside on the same support. In exemplary embodiments, lab-on-a-chip-like devices, available for example from Caliper and Fluidigm, are used as reaction vessels for the rapid PCR. It will be recognized that a variety of reaction vessels are available in the art and fall within the scope of the present teachings.

[0160] In exemplary embodiment, rapid PCR includes performing simplex PCR, or a single assay for detecting a single target.[00161 J In exemplary embodiments, rapid PCR includes performing multiplex PCR, or an assay for detecting two or more targets in a single reaction vessel.

[0162] The rapid PCRs disclosed herein involve a thermal cycling including an initial denaturing step followed by a repetitive series of temperature cycles designed to allow template denaturation, primer annealing, and extension of the annealed primers by a polymerase. Thermal cycling can also include additional temperature shifts. The number of cycles used during the rapid PCR can depend on many factors, including the primers used, the amount of sample DNA present, and the thermal cycling conditions. The number of cycles to be used in the methods disclosed herein can be readily determined by one skilled in the art using routine experimentation. Optionally, a final extension step can be added after the completion of thermal cycling to ensure synthesis of all amplification products.

[0163] In exemplary embodiments, the rapid PCR includes at least one or more of the following: performing a pre-PCR read of the crude nucleic acid sample, incubated in the PCR mixture, for about 10-15 seconds; performing an initial denaturation of the incubated crude nucleic acid sample for about 5-10 minutes to form a denatured nucleic acid sample; performinga second denaturation of the denatured nucleic acid sample for about one second followed by annealing and extension for about 10-15 seconds to form amplicons; repeating the performing of the second denaturation and the annealing and extension between one to forty times; and performing a post-PCR read of the amplicons for about 10 seconds. In exemplary embodiments, the pre-PCR read is performed at a temperature from 55°C to 65°C. In exemplary embodiments, the initial denaturation of the rapid PCR is performed at a temperature from 85°C to 100°C, or any temperature or temperature range falling therein. In exemplary embodiments, the second denaturation of the rapid PCR is performed at a temperature from 85°C to 100°C, or any temperature or temperature range falling therein. In exemplary embodiments, the post-PCR read of the rapid PCR is performed at a temperature from 55°C to 65°C, or any temperature or temperature range falling therein. In exemplary embodiments, the post-PCR read is an end-point PCR read. In yet other exemplary embodiments, subsampling is performed between the pre-PCR read and the end-point PCR read.

[0164] In exemplary embodiments, the performing of the rapid PCR includes using at least one probe and at least one reporter dye.

[0165] In exemplary embodiments, the rapid PCR includes at least one or more of the following: performing a pre-PCR read of the crude nucleic acid sample, incubated in the PCR mixture, at a temperature of about 60°C; performing an initial denaturation of the incubated crude nucleic acid sample at a temperature of about 95°C; performing a second denaturation of the denatured nucleic acid sample, followed by an annealing and extension of the denatured nucleic acid sample, at a temperature of about 95°C to form amplicons; repeating the performing of the second denaturation and the annealing and extension at a temperature of about 95°C; and performing a post-PCR read of the amplicons at a temperature of about 60°C.

[0166] In exemplary embodiments, the rapid PCR is a simplex PCR. In exemplary embodiments, the rapid PCR is a multiplex PCR. The term “simplex” or “simplex PCR” as used herein refers to an assay that provides for amplification of a single product or target within a reaction vessel. The product is primed using a distinct primer pair. A simplex reaction can further include a labeled probe specific for the amplified product or target, wherein the probe is detectably labeled with a detectable moiety, such as a fluorescent dye.

[0167] In exemplary embodiments, the rapid PCR is a multiplex PCR. The term “multiplex” or “multiplex PCR” as used herein refers to an assay that provides for simultaneous amplification of two or more products or targets within the same reaction vessel. Each product or target is primed using a distinct primer pair. A multiplex reaction can also include labeled probes specific to each product or target, wherein the probes are detectably labeled with different detectable moieties. In exemplary embodiments, the rapid PCR is a multiplex PCR that includes those in which: (i) a multiplicity of targets that are amplified in a single sample; two or more targets in one sample or (ii) multiple samples are simultaneously amplified; two targets in two different samples within a single reaction at substantially the same time.

[0168] In exemplary embodiments, the rapid PCR is a multiplex PCR wherein the number of targets amplified are up to as many as 2 targets, 5 targets, 10 targets, 25 targets, 50 targets, 100 targets, 1000 targets, or 5000 targets, and so on, including all numbers in between. In exemplary embodiments, one of the multiplex PCR’s targets is an endogenous or an exogenous internal positive control for amplification. In exemplary embodiments, the number of targets amplified and / or detected with the multiplex PCR are up to 25 targets, 10 targets, 8 targets, 6 targets, 5 targets, 4 targets, 3 targets, or 2 targets. In exemplary embodiments, a multiplicity of targets is simultaneously amplified and detected during the multiplex PCR. In exemplary embodiments, a multiplicity of targets is amplified and detected in the same reaction vessel. In exemplary embodiments, various TaqMan™ probe reporter dye and passive dye options are combined and used in the multiplex PCR. For example, TaqMan™ probes with FAM™, VIC™, and ABY™ reporter dyes in combination with a PCR composition containing ROX1Mpassive reference dye can used for a 3-plex multiplex PCR amplification and detection assay. For another example, TaqMan™ probes with FAM™, VIC™, ABY™, and JUN™ reporter dyes in combination with a PCR composition containing MUSTANG PURPLE™ passive reference dye can be used for a 4-plex multiplex PCR amplification and detection assay. In exemplary embodiments, nucleic acid synthesis (such as a nucleic acid amplification reaction or a PCR) and nucleic acid detection (e.g., of an amplicon) can occur simultaneously during the multiplex PCR. Accordingly, in exemplary embodiments, nucleic acid synthesis and nucleic acid detection occur in the same reaction vessel.

[0169] In exemplary embodiments, the rapid PCR is a hot start PCR. The hot start PCR can include the use of manual techniques, barriers, reversible polymerase inactivation, and / or specially-designed hairpin primers. In general, manual hot start PCR methods usually, though not always, require the researcher to withhold a critical component, usually magnesium or the polymerase, until the reaction has been heated. The withheld component then is added to initiate the reaction. A second method uses a physical barrier (e.g., wax) to separate a critical component from the template and primers. U.S. Pat. No. 5,565,339 describes using a wax barrier to separate the various PCR reagents from each other in a test tube. U.S. Pat. No. 5,413,924 describes using a paraffin wax bead to sequester the DNA polymerase.

[0170] In exemplary embodiments, the hot start PCR includes reversible polymerase inactivation. Reversible polymerase inactivation can involve reacting a polymerase with an antibody or an oligonucleotide aptamer that binds to the polymerase's nucleotide binding domain, rendering the polymerase inactive. For example, a monoclonal antibody to Taq polymerase, such as the anti-Taq DNA polymerase antibody available from Sigma, is introduced into the reaction mixture. Upon heating, the compound dissociates from the polymerase, restoring enzyme activity. In another example, U.S. Pat. No. 5,677,152 describes a method in which the DNA polymerase is chemically modified to ensure that it only becomes active at elevated temperatures.

[0171] In exemplary embodiments, the hot start PCR includes primers that will selfanneal to form specific hairpin structures. The hairpin primers will not be able to anneal to the target nucleic acid while in the hairpin conformation. The hairpin primers will remain in a hairpin conformation until heated to a denaturation temperature. In exemplary embodiments, the primers include a hairpin structure possessing a single-strand extension. In these embodiments, the hairpin structure itself resembles a primer annealed to a template and can result in strand extension.

[0172] Various other hot start components or mechanisms, in addition to those described above, are also well known to those of ordinary skill in the art and can be used in the methods disclosed herein. In exemplary embodiments, the hot start PCR includes at least two different hot start mechanisms that inhibit or substantially inhibit the polymerase activity of a nucleic acidpolymerase under a first condition (such as at a lower temperature) and allow polymerase activation under a second condition (such as at a higher temperature). Such hot start mechanisms include, but are not limited to, those described above including antibodies or combinations of antibodies that block DNA polymerase activity at lower temperatures, oligonucleotides that block DNA polymerase activity at lower temperatures, reversible chemical modifications of the DNA polymerase that dissociate at elevated temperatures, amino acid modifications of the DNA polymerase that provide reduced activity at lower temperatures, fusion proteins that include hyperstable DNA binding domains and topoisomerase, temperature dependent ligands that inhibit the DNA polymerase, single stranded binding proteins that sequester primers at lower temperatures, modified primers or modified dNTPs.

[0173] The rapid PCRs disclosed herein can be performed on “standard” PCR instrumentation, e.g., Applied Biosystems™ 7900HT, 7500, and 7300 standard PCR systems, or on “Fast” PCR instrumentation, e.g., Applied Biosystems™ StepOne, StepOne Plus, 7500 and 7900HT Fast Real-Time PCR systems. In exemplary embodiments, the rapid PCR includes performing at least one thermal cycling with an instrument selected from, but not limited to, a GeneAmp™PCR System 9700, 9600, 2700 or 2400 thermocycler, an Applied Biosystems™ ViiA™ 7 Real-Time PCR System, a QuantStudio™ 12K Flex Real-Time PCR System, a QuantStudio™ Dx Real-Time PCR System, a QuantStudio™ 6 Flex Real-Time PCR System, a QuantStudio™ 7 Flex Real-Time PCR System, a QuantStudio™ 3 Real-Time PCR System, or a QuantStudio™ 5 Real-Time PCR System. Other examples of spectrophotometric thermal cyclers for use in the methods include, but are not limited to, Bio-Rad ICycler IQ™, Cepheid SmartCycler™ II, Corbett Research Rotor-Gene 3000, Idaho Technologies R.A.P.I.D.™, MJ Research Chromo 4™, Roche Applied Science LightCycler™, Roche Applied Science LightCycler™2.0, Stratagene Mx3000P™, and Stratagene Mx4000™.

[0174] In exemplary embodiments, the method includes determining a genotype of the nucleic acid sample using an amplification product of the rapid PCR. In exemplary embodiments, the method includes determining a copy number of a target polynucleotide sequence in the nucleic acid sample using an amplification product of the rapid PCR.

[0175] In exemplary embodiments, the method includes isolating the amplified or synthesized nucleic acid fragments created during the rapid PCR for further use or characterization. This step can be accomplished by separation of the amplified or synthesized nucleic acid fragments by size or by any physical or biochemical means including gel electrophoresis, capillary electrophoresis, chromatography (including sizing, affinity and immunochromatography), density gradient centrifugation and immunoadsorption. An exemplary method is separation of nucleic acid fragments by gel electrophoresis, which provides a rapid and highly reproducible means of sensitive separation of a multitude of nucleic acid fragments, and permits direct, simultaneous comparison of the fragments in several samples of nucleic acids.

[0176] In exemplary embodiments, the method includes removing one or more of the amplified or synthesized nucleic acid fragments created during the rapid PCR from a gel by chemical extraction, electroelution, or physical excision. The isolated unique nucleic acid fragments can then be inserted into standard vectors, including expression vectors, suitable for transfection or transformation of a variety of prokaryotic (bacterial) or eukaryotic (yeast, plant or animal including human and other mammalian) cells. Alternatively, nucleic acids produced by the rapid PCRs disclosed herein can be characterized, for example, by sequencing (i.e., determining the nucleotide sequence of the nucleic acid fragments), by methods described below and others that are standard in the art (see, e.g., U.S. Pat. Nos. 4,962,022 and 5,498,523, which are directed to methods of DNA sequencing). Classical sequencing methods can also be employed, such as the Sanger chain termination method (Sanger, F., et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) and the Maxam and Gilbert chemical cleavage method (Maxam, A. M. and Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977)).

[0177] In exemplary embodiments, the amplified or synthesized nucleic acid fragments created during the rapid PCR are characterized by next generation sequencing. As used herein, the term “next generation sequencing” or “NGS” generally refers to high throughput sequencing technologies, including, but not limited to, massively parallel signature sequencing, high throughput sequencing, sequencing by ligation e.g., SOLiD sequencing), proton ion semiconductor sequencing, DNA nanoball sequencing, single molecule sequencing, and nanopore sequencing.

[0178] Another aspect of the present disclosure is a method of detecting a disease in a subject, the method including at least one or more of: subjecting a reaction mixture consisting of a polymerase chain reaction (PCR) mixture and a crude nucleic acid sample from the subject to rapid PCR by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least one second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and determining a presence and / or amount of at least one target nucleic acid sequence in the crude nucleic acid sample, the at least one target nucleic acid sequence being a biomarker of the disease.

[0179] The PCR mixture used in the method can be any PCR mixture discussed in the present disclosure. In exemplary embodiments, the PCR mixture includes a hot-start component. The hot-start component can inhibit polymerase activity of a polymerase in the PCR mixture and can include at least one or more of an oligonucleotide, a temperature-dependent ligand, an aptamer, and antibodies specific to the polymerase and / or an agent that mediates a reversible chemical modification of the polymerase. The crude nucleic acid sample used in the method can be any crude nucleic acid sample discussed in the present disclosure and can be derived from any source discussed in the present disclosure. The amplification process can be any rapid PCR procedure discussed in the present disclosure.

[0180] The methods disclosed herein are effective at reducing PCR run times as compared to an equivalent PCR run involving the use of commercially available master mixes and sample preparation procedures. This was particularly the case when the nucleic acid sample added to the PCR was a crude sample and / or the target sequence was present at a low copy number. The methods’ reductions of PCR run times can be demonstrated by lower Ct values. In exemplary embodiments, the method produces a Ct value that is at least 10 fold, at least 50 fold, at least 100 fold, at least 500 fold, or at least 1000 fold lower when compared to an equivalent PCR run involving the use of commercially available master mixes and sample preparation procedures.

[0181] In exemplary embodiments, a Ct value is determined using a derivative of a PCR curve. For example, a first, second, or nth order derivative method can be performed on a PCR curve in order to determine a Ct value. In exemplary embodiments, a characteristic of a derivative can be used in the determination of a Ct value. Such characteristics can include, but are not limited by, a positive inflection of a second derivative, a negative inflection of a second derivative, a zero crossing of the second derivative, or a positive inflection of a first derivative. In exemplary embodiments, a Ct value can be determined using a thresholding and baselining method. For example, an upper bound to an exponential phase of a PCR curve can be established using a derivative method, while a baseline for a PCR curve can be determined to establish a lower bound to an exponential phase of a PCR curve. From the upper and lower bound of a PCR curve, a threshold value can be established from which a Ct value is determined. Other methods for the determination of a Ct value known in the art can be used, for example, but not limited to, the various embodiments of a fit point method and of a sigmoidal method (See, e.g., U.S. Patent Nos. 6,303,305; 6,503,720; 6,783,934, 7,228,237 and U.S. Application No. 2004 / 0096819; the disclosures of which are herein incorporated by reference in their entireties).[00182J The methods provided herein offer advantages over standard or traditional genotyping methods. In exemplary embodiments, such advantages include, but are not limited, to any of the following: (i) can be used to quickly amplify nucleic acids (e.g., by real time qPCR) directly from unprocessed samples (e.g., can amplify targets directly from buccal samples, saliva and whole blood) without the need to subject the samples to a lysis buffer or nucleic acid extraction solution; (ii) improves the accuracy of copy number variation results; (iii) provides increased tolerance to various PCR inhibitors; (iv) provides increased specificity and sensitivity; (v) can be used with fast thermal cycling protocols for quicker read-outs; (vi) allows for the capability to multiplex in a single reaction at substantially the same time; and / or (vii) allows for the capability of genotyping in a mobile setting (i.e., outside of a specialized lab infrastructure).

[0183] In exemplary embodiments, the method is completed in 30 minutes or less. In exemplary embodiments, the method is completed in about 27 minutes or less, or about 25 minutes or less. In exemplary embodiments, the method is completed in about 23 minutes.

[0184] In exemplary embodiments, the method excludes subjecting the crude nucleic acid sample to a lysing solution.

[0185] In exemplary embodiments, the method excludes subjecting the crude nucleic acid sample to a nucleic acid extraction solution.

[0186] In exemplary embodiments, the method is performed in the absence of a lysing solution and / or a nucleic acid extraction solution.

[0187] FIG. 2 is a conventional workflow for sample preparation and PCR.

[0188] Referring to FIG. 2, according to a conventional workflow includes preparing the samples by subjecting a nucleic acid sample to a lysis solution.

[0189] Then, if the nucleic acid sample contains low copy numbers, the lysed nucleic acid sample may be pre-amplified by subjecting the lysed sample to a pre-amplification mix, and then diluting the preamplified nucleic acid sample.

[0190] The lysed and / or pre-amplified nucleic acid sample is then subjected to PCR, and the results are analyzed.EXAMPLES

[0191] Conventional Example 1 was prepared according to a conventional workflow.

[0192] Preparation of Conventional Example 1 :

[0193] A crude nucleic acid sample was obtained by rotating and firmly brushing a dry swab using about 20 strokes throughout the inside of the cheek.

[0194] The crude nucleic acid sample was then lysed using the DNA Extract All Reagents Kit (manufactured by Thermo Fisher Scientific). In particular, the dry swab was immersed into 400 pL of lysis solution in microcentrifuge tube, and rotated about 5 times to mix the lysis solution and the crude nucleic acid sample.

[0195] Excess sample and / or solution was removed from the swab by lifting the swab above the lysis solution, and then pressing the swab against the inside of the tube to squeeze out its contents.

[0196] The tube was then briefly centrifuged.

[0197] The lysed nucleic acid sample within the tube was incubated at 95 °C for 3 minutes.

[0198] 400 pL of DNA Stabilizing Solution was added to the tube, and mixed well.

[0199] Next, a PCR mixture was prepared, and the stabilized nucleic acid sample was added to the PCR mixture.

[0200] Then, PCR was performed according to a conventional genotyping protocol, and the results were analyzed.

[0201] FIG. 3A is an allelic discrimination plot of the amplified product of the Comparative Example 1.

[0202] Example 1 according to exemplary embodiments was prepared as follows.

[0203] A crude nucleic acid sample was obtained by rotating and firmly brushing a puritan dry foam swab using about 10 strokes throughout the inside of the cheek.

[0204] Next, the crude nucleic acid sample was added directly to 250pL of a PCR mixture (TaqPath™ ProAmp1''1Multiplex master mix with a TaqMan™ probe). The buccal swab was swirled in the PCR mixture for 10 seconds.

[0205] Then, PCR was performed on a 10 pL aliquot of the mixture according to the following rapid PCR protocol:1. Pre-PCR read at 60°C for 10 second.2. Initial denaturation at 95°C for 5 minutes.3. Sub sampling:3A. Denaturation at 95°C for 1 second followed by annealing and extension for 3 seconds. Four cycles without data collection.3B. Denaturation at 95°C for 1 second followed by annealing and extension for 10 seconds. 1 cycle with real-time data collection.4. Repeat step 3 an additional seven times.5. Post-PCR read at 60°C for 10 seconds.Accordingly, 40 rapid PCR cycles were performed, with real-time data collection occurring during 8 of the cycles. The instrument run time was 23 minutes.

[0206] The results were analyzed.

[0207] FIG. 3B is an allelic discrimination plot of the amplified product of Example 1.

[0208] Comparing FIG. 3B with FIG. 3A, although the signal intensity is lower for Example 1, the data quality is comparable and generates equivalent genotyping results.

[0209] In exemplary embodiments, a thermal cycling and data collection method are used to enable a shortened instrument run time, by conducting a plurality of amplification cycles without data collection followed by at least one amplification cycle with data collection.

[0210] In exemplary embodiments, a thermal cycling and data collection method are used to enable a shortened instrument run time, by conducting a plurality of amplification cycles with a shorter denaturation time and / or a shorter annealing and extension time and without data collection, followed by at least one amplification cycle with the shorter denaturation time and / or a longer annealing and extension time (than the earlier amplification cycles) and with data collection.

[0211] Disclosed, in a first aspect, is a method of genotyping a crude nucleic acid sample, the method comprising: subjecting the crude nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the crude nucleic acid sample from a source; performing rapid PCR on the crude nucleic acid sample by subjecting the crude nucleic acidsample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and analyzing results from the rapid PCR to determine a genotype of the crude nucleic acid sample.

[0212] In the first aspect, the rapid PCR can be performed in less than 30 minutes.

[0213] In the first aspect, the rapid PCR can be performed in about 20 minutes.

[0214] In the first aspect, the performing rapid PCR can further comprise: incubating the crude nucleic acid sample in the PCR mixture for a set period of time, performing a pre-PCR read of the incubated crude nucleic acid sample for about 10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

[0215] In the first aspect, the first plurality of amplification cycles can include four amplification cycles where each cycle includes performing the second denaturation for about one second followed by annealing and extension for 3 seconds, and the at least one second amplification cycle includes performing the second denaturation for about one second followed by annealing and extension for 10 seconds.

[0216] In the first aspect, the performing rapid PCR on the crude nucleic acid sample can include subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle 8 times.

[0217] In the first aspect, the pre-PCR read and the post-PCR read can be performed at a temperature from 55°C to 65°C.

[0218] In the first aspect, the initial denaturation and the second denaturation can be performed at a temperature from 85°C to 100°C.

[0219] In the first aspect, the pre-PCR read and the post-PCR read can be performed at a temperature of about 60°C, and the initial denaturation and the second denaturation are performed at a temperature of about 95 °C.

[0220] Disclosed in a second aspect is a method of detecting a disease in a subject, comprising: subjecting a reaction mixture consisting essentially of a polymerase chain reaction (PCR) mixture and a crude nucleic acid sample from the subject to rapid PCR by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least one second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and determining a presence and / or amount of at least one target nucleic acid sequence in the crude nucleic acid sample, the at least one target nucleic acid sequence being a biomarker of the disease.

[0221] In the first and second aspects, the PCR mixture can include a hot-start component.

[0222] In the first and second aspects, the hot-start component can inhibit polymerase activity of a polymerase in the PCR mixture and can include at least one or more of an oligonucleotide, a temperature-dependent ligand, an aptamer, and antibodies specific to the polymerase and / or an agent that mediates a reversible chemical modification of the polymerase.

[0223] In the first and second aspects, the method can be performed in the absence of a lysing solution and / or a nucleic acid extraction solution.

[0224] In the first and second aspects, the source can be a buccal cavity.

[0225] In the first and second aspects, the crude nucleic acid sample can be obtained using a swab, the swab being at least one selected from a dry swab, a dry foam swab, a flocked swab and a moisten or wet swab.

[0226] In the first and second aspects, the swab can be formed of foam, cotton, flock, rayon, polyester, calcium alginate or any combination thereof.

[0227] In the first and second aspects, the swab is not subjected to a transport medium prior to the crude nucleic acid sample being subjected to the PCR mixture, or in the reaction mixture.

[0228] In the first and second aspects, the source can be a buccal cavity, and the method further comprises swabbing the buccal cavity to obtain the crude nucleic acid sample.

[0229] In the first and second aspects, the source can be a hair follicle.

[0230] In the first and second aspects, the crude nucleic acid sample can be obtained at least 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.

[0231] In the first and second aspects, the crude nucleic acid sample can be obtained within 24 to 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.

[0232] In the first and second aspects, the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, can be an undiluted sample.

[0233] In the first and second aspects, the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, can be an unpurified sample.

[0234] In the first and second aspects, the subjecting of the reaction mixture to the rapid PCR can include performing one selected from polymerase chain reaction (PCR), real-time PCR, quantitative PCR, multiplex PCR, methylation-specific PCR and endpoint PCR.

[0235] In the first and second aspects, the subjecting of the reaction mixture to the rapid PCR can include: incubating the crude nucleic acid sample in the PCR mixture for a set period oftime, performing a pre-PCR read of the incubated crude nucleic acid sample for about 10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

[0236] In the first and second aspects, the post-PCR read can be an end-point PCR read.

[0237] In the first and second aspects, the PCR mixture or the reaction mixture can contain deoxynucleotide triphosphates, magnesium ions, potassium ions, a buffer solution, at least one primer pair specific for nucleotides in a target nucleic acid in the crude nucleic acid sample, at least one probe specific for nucleotides in the target nucleic acid, and at least one thermostable polymerase, wherein the at least one probe includes a detectable label.

[0238] In the first and second aspects, the target nucleic acid can be a biomarker for a disease.

[0239] In the first and second aspects, the at least one thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, Platinum Taq DNA polymerase, Platinum II Taq Hot-Start DNA polymerase, AmpliTaq DNA polymerase, Invitrogen Taq DNA, Dream Taq DNA polymerase and mutants or derivatives thereof having DNA polymerase activity.

[0240] In the first and second aspects, the PCR mixture can contain a PCR inhibitor blocking agent.

[0241] In the first and second aspects, the PCR inhibitor blocking agent can be selected from the group consisting of an albumin, a gelatin, and a combination thereof.

[0242] In the first and second aspects, the performing the rapid PCR, or the determining the presence and / or amount of at least one target nucleic acid sequence, can include using at least one probe and at least one reporter dye.

[0243] In the first and second aspects, the method can be used to perform a genotyping assay.

[0244] The disclosed methods are applicable to genetic analysis, and more specifically, genotyping. However, embodiments are not limited thereto.

[0245] According to exemplary embodiments, methods of genotyping using rapid PCR are disclosed wherein the overall instrument run time (of 30 minutes or less, or preferably 23 minutes) is less than that observed in the conventional genotyping methods (which are typically at least 40 minutes or more).

[0246] Furthermore, in conventional genotyping protocols, real-time amplification data is obtained as a part of genotyping analysis. This feature allows for data traces to be visualized in a 2D cluster plot and improves data quality. Without collecting real-time amplification data during PCR, genotyping can be accomplished via endpoint analysis. However, the lack od data traces can lead to higher rates of undetermined calls.

[0247] Subsampling (i.e., collecting data at the at least one amplification cycle with the shorter denaturation time and / or a longer annealing and extension time) according to exemplary embodiments enables a user to obtain real-time amplification data in combination with significantly shorter amplification cycles during non-data collection cycles.

[0248] Thus, the inventors have surprisingly found that even faster genotyping can be achieved with the shorten instrument run time combined with the direct amplification of a crude nucleic acid sample than observed in conventional genotyping protocols. Thus, using the disclosed genotyping methods would allow faster analysis and testing, especially in settings thatrequire testing a large number of samples (e g., clinical, animal or livestock testing, etc.) with a quick turnaround time.

[0249] As those skilled in the art will appreciate, numerous changes and modifications can be made to the various embodiments disclosed herein without departing from the spirit of the present disclosure. It is intended that all such variations fall within the scope of this disclosure.

[0250] All of the methods and mixtures disclosed herein, can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of these teachings have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations can be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the concept and scope of this disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein, while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of this disclosure.

Claims

CLAIMSWhat is claimed is:

1. A method of genotyping a crude nucleic acid sample, the method comprising: subjecting the crude nucleic acid sample to a polymerase chain reaction (PCR) mixture directly after obtaining the crude nucleic acid sample from a source; performing rapid PCR on the crude nucleic acid sample by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least second amplification cycle includes performing denaturation followed by annealing and extension while simultaneously collecting amplification data; and analyzing results from the rapid PCR to determine a genotype of the crude nucleic acid sample.

2. The method of claim 1, wherein the rapid PCR is performed in less than 30 minutes.

3. The method of clam 1, wherein the rapid PCR is performed in about 20 minutes.

4. The method of any one of claims 1-3, wherein the performing rapid PCR further comprises: incubating the crude nucleic acid sample in the PCR mixture for a set period of time, performing a pre-PCR read of the incubated crude nucleic acid sample for about 10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation for about one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

5. The method of any one of claims 1-4, wherein the first plurality of amplification cycles includes four amplification cycles where each cycle includes performing the second denaturation for about one second followed by annealing and extension for 3 seconds, and the at least one second amplification cycle includes performing the second denaturation for about one second followed by annealing and extension for 10 seconds.

6. The method of any one of claims 1-5, wherein the performing rapid PCR on the crude nucleic acid sample includes subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle 8 times.

7. The method of any one of claims 1-6, wherein the pre-PCR read and the post- PCR read are performed at a temperature from 55°C to 65°C.

8. The method of any one of claims 1-7, wherein the initial denaturation and the second denaturation are performed at a temperature from 85°C to 100°C.

9. The method of any one of claims 1-8, wherein the pre-PCR read and the post- PCR read are performed at a temperature of about 60°C, and the initial denaturation and the second denaturation are performed at a temperature of about 95°C.

10. A method of detecting a disease in a subject, comprising: subjecting a reaction mixture consisting essentially of a polymerase chain reaction (PCR) mixture and a crude nucleic acid sample from the subject to rapid PCR by subjecting the crude nucleic acid sample to a first plurality of amplification cycles and followed by at least one second amplification cycle, wherein each cycle of the first plurality of amplification cycles includes performing denaturation followed by annealing and extension without collecting amplification data, and wherein the at least one second amplification cycle includes performingdenaturation followed by annealing and extension while simultaneously collecting amplification data; and determining a presence and / or amount of at least one target nucleic acid sequence in the crude nucleic acid sample, the at least one target nucleic acid sequence being a biomarker of the disease.

11. The method of any one of claims 1-10, wherein the PCR mixture includes a hot- start component.

12. The method of claim 11, wherein the hot-start component inhibits polymerase activity of a polymerase in the PCR mixture and includes at least one or more of an oligonucleotide, a temperature-dependent ligand, an aptamer, and antibodies specific to the polymerase and / or an agent that mediates a reversible chemical modification of the polymerase.

13. The method of any one of claims 1-12, wherein the method is performed in the absence of a lysing solution and / or a nucleic acid extraction solution.

14. The method of any one of claims 1-13, wherein the source is a buccal cavity.

15. The method of any one of claims 1-14, wherein the crude nucleic acid sample is obtained using a swab, the swab being at least one selected from a dry swab, a dry foam swab, a flocked swab and a moisten or wet swab.

16. The method of claim 14, wherein the swab is formed of foam, cotton, flock, rayon, polyester, calcium alginate or any combination thereof.

17. The method of claim 14, wherein the swab is not subjected to a transport medium prior to the crude nucleic acid sample being subjected to the PCR mixture, or in the reaction mixture.

18. The method of any one of claims 1-17, wherein the source is a buccal cavity, and the method further comprises swabbing the buccal cavity to obtain the crude nucleic acid sample.

19. The method of any one of claims 1-13 and 15-17, wherein the source is a hair follicle.

20. The method of any one of claims 1-19, wherein the crude nucleic acid sample is obtained at least 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.21 . The method of any one of claims 1-20, wherein the crude nucleic acid sample is obtained within 24 to 120 hours prior to being subjected to the PCR mixture, or being in the reaction mixture.

22. The method of any one of claims 1-21, wherein the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, is an undiluted sample.

23. The method of any one of claims 1-21, wherein the crude nucleic acid sample subjected to the PCR mixture, or in the reaction mixture, is an unpurified sample.

24. The method of any one of claims 10-23, wherein the subjecting of the reaction mixture to the rapid PCR includes performing one selected from polymerase chain reaction (PCR). real-time PCR, quantitative PCR, multiplex PCR, methylation-specific PCR and endpoint PCR.

25. Tire method of any one of claims 10-24, wherein the subjecting of the reaction mixture to the rapid PCR includes: incubating the crude nucleic acid sample in the PCR mixture for a set period of time, performing a pre-PCR read of the incubated crude nucleic acid sample for about10 seconds, performing an initial denaturation of the incubated crude nucleic acid sample for about five minutes, before subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle, and performing a post-PCR read for about 10 seconds of amplicons generated as a result of subjecting the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle; and the subjecting of the crude nucleic acid to the first plurality of amplification cycles and the at least one second amplification cycle comprises performing a second denaturation forabout one second followed by annealing and extension for about 3-10 seconds, and repeating the performing of the second denaturation and the annealing and extension between one to forty times.

26. The method of any one of claims 4-25, wherein the post-PCR read is an end-point PCR read.

27. The method of any one of claims 1-26, wherein the PCR mixture or the reaction mixture contains deoxynucleotide triphosphates, magnesium ions, potassium ions, a buffer solution, at least one primer pair specific for nucleotides in a target nucleic acid in the crude nucleic acid sample, at least one probe specific for nucleotides in the target nucleic acid, and at least one thermostable polymerase, wherein the at least one probe includes a detectable label.

28. The method of claim 27, wherein the target nucleic acid is a biomarker for a disease.

29. The method of claim 27 or 28, wherein the at least one thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENT™ DNA polymerase, DEEP VENT™ DNA polymerase, Platinum Taq DNA polymerase, Platinum II Taq Hot-Start DNA polymerase, AmpliTaq DNA polymerase, Invitrogen Taq DNA, Dream Taq DNA polymerase and mutants or derivatives thereof having DNA polymerase activity.

30. The method of any one of claims 1 -29, wherein the PCR mixture contains a PCR inhibitor blocking agent.31 . The method of claim 30, wherein the PCR inhibitor blocking agent is selected from the group consisting of an albumin, a gelatin, and a combination thereof.

32. The method of any one of claims 1-31, wherein the performing the rapid PCR, or the determining the presence and / or amount of at least one target nucleic acid sequence, includes using at least one probe and at least one reporter dye.

33. The method of any one of claims 1-32 being used to perform a genotyping assay.

Images