WO2025007089A1 - Compositions, méthodes et kits de synthèse et d'amplification d'acides nucléiques - Google Patents

Compositions, méthodes et kits de synthèse et d'amplification d'acides nucléiques Download PDF

Info

Publication number
WO2025007089A1
WO2025007089A1 PCT/US2024/036306 US2024036306W WO2025007089A1 WO 2025007089 A1 WO2025007089 A1 WO 2025007089A1 US 2024036306 W US2024036306 W US 2024036306W WO 2025007089 A1 WO2025007089 A1 WO 2025007089A1
Authority
WO
WIPO (PCT)
Prior art keywords
qpcr
nucleic acid
composition
antifoam
sulfate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/036306
Other languages
English (en)
Inventor
Jennifer Berkman
Ignas URBONAS
Daniela Cruz ABREU
Chengjing LIU
Chia An YEN
Kimberlie Soohoo ONG
Wei Wei
Maria Cinta GOMEZ SILVAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Thermo Fisher Scientific Baltics UAB
Life Technologies Corp
Original Assignee
Thermo Fisher Scientific Baltics UAB
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thermo Fisher Scientific Baltics UAB, Life Technologies Corp filed Critical Thermo Fisher Scientific Baltics UAB
Publication of WO2025007089A1 publication Critical patent/WO2025007089A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • compositions, methods and kits for the synthesis of nucleic acids relate to compositions, methods and kits for the synthesis of nucleic acids. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a real-time/quantitative polymerase chain reaction (qPCR) procedure, such as, for example, a one-step qPCR or a one-step reverse transcription qPCR (RT-qPCR) procedure, including one or more agents used to increase stability.
  • qPCR real-time/quantitative polymerase chain reaction
  • RT-qPCR reverse transcription qPCR
  • RNA reverse transcription of ribonucleic acid
  • PCR polymerase chain reaction
  • RT-qPCR Quantitative or “real-time” reverse transcription polymerase chain reaction
  • mRNA messenger RNA
  • qPCR real-time PCR reaction
  • the amount of amplification product is measured in each PCR cycle using fluorescence from fluorescent labels.
  • RT-qPCR has a variety of applications, including gene expression analysis, RNA interference (RNAi) validation, microarray validation, pathogen detection, genetic testing, and disease research.
  • RNAi RNA interference
  • RT-PCR and RT-qPCR typically involve two separate molecular syntheses: (i) the synthesis of cDNA from an RNA template; and (ii) the replication of newly synthesized cDNA through PCR amplification.
  • RT-PCR and RT-qPCR can be performed by one-step (or coupled) RT-PCR and RT-qPCR methods using two or more enzymes, in which at least two separate enzymes (e.g., a reverse transcriptase and a polymerase) are employed for initial cDNA synthesis and subsequent amplification, respectively.
  • samples from which nucleic acids are extracted often contain additional compounds that are inhibitory to PCR.
  • Humic acid in soil and feces, hematin in blood, immunoglobin G in serum, and various blood anticoagulants, like heparin and citrate, are all examples of such inhibitors.
  • Such inhibitors may not be completely removed during the nucleic acid extraction and purification process, thus negatively impacting downstream PCR amplification, as reflected by an increase in Ct (i.e., threshold cycle) and decrease in dRn (z.e., difference in normalized reporter signal) when assayed by real time PCR.
  • a high Ct coupled with low dRn usually indicates low target nucleic acid concentration in reactions for quantitative PCR (qPCR) and reverse transcriptase-qPCR (RT-qPCR) applications.
  • qPCR quantitative PCR
  • RT-qPCR reverse transcriptase-qPCR
  • a reaction that exhibits reduced or no amplification indicates that the target nucleic acid is absent, or present in such small amounts that it is not detectable.
  • a reaction that contains detectable amounts of target, but is inhibited by the presence of qPCR inhibitors may show an artificially high threshold cycle (Ct) and low fluorescence signal (dRn), which can lead the user to believe that the amount of target nucleic acid is less than the actual amount present. If the level of inhibition is severe enough, the reaction may fail to amplify completely, thus leading to a false-negative result.
  • Current one-step RT-qPCR methods have limited multiplex ability, limited at least in part by the benchtop stability and the reaction efficiency of the process.
  • a one-step RT-qPCR system in the form of a generalized ready -to-use composition, which exhibits high sensitivity, is not restricted by the amount of sample, reduces the amount of practitioner manipulation, minimizes the risks of contamination, minimizes the expense of reagents, maximizes multiplex validation, and/or maximizes the amount of nucleic acid end product, is needed in the art.
  • a method to increase or maximize the stability of assembled reactions and the reaction efficiency, especially when multiple targets are present in the sample is necessary to ensure accurate results.
  • compositions, methods and kits useful for the synthesis of nucleic acids. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a one-step qPCR or RT-qPCR procedure using one or more reverse transcriptases, such as Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, in combination with one or more DNA polymerases, such as DNA polymerase from Thermophilus aquations (Taq).
  • M-MLV Moloney Murine Leukemia Virus
  • DNA polymerases such as DNA polymerase from Thermophilus aquations (Taq).
  • the compositions further comprise one or more agents used to increase stability of assembled reactions of a polymerase chain reaction.
  • stabilizing agents can include, for example, an RNA aptamer and/or a salt.
  • the present disclosure thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of target sequences which can be used for a variety of industrial, medical, forensic and diagnostic purposes.
  • the embodiments disclosed herein are especially useful for the rapid amplification and detection of viral genes, including both RNA and DNA targets.
  • compositions comprising at least one active DNA polymerase and at least one active reverse transcriptase (RT).
  • compositions further comprise at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction.
  • the reverse transcriptases are thermostable.
  • the thermostable reverse transcriptases can be M-MLV reverse transcriptases, mutants, variants, or derivatives thereof.
  • the M-MLV RTs can comprise one or more mutations. Such mutations can include, for example: Y64, R116, DI 24, Hl 26, Y133, KI 52, Q190, T197, H204, V223, M289, T306, or F309.
  • the concentration of the reverse transcriptase(s) is between about 0.5 U/pL to about 5 U/pL.
  • the DNA polymerases are thermostable DNA polymerases.
  • the thermostable DNA polymerases can be Taq DNA polymerases, mutants, variants, or derivatives thereof, such as, for example, AmpliTaq® Fast DNA polymerase or a mutant, variant, or derivative thereof.
  • the concentration of the DNA polymerase(s) is between about 0.005 U/pL to about 3.0 U/pL.
  • the present composition includes qPCR inhibitor blocking agents of the present compositions include a neutral compound with cationic group lacking hydrogen. In some embodiments, the qPCR inhibitor blocking agents of the present compositions include a nonionic polyoxyethylene surfactant.
  • the present compositions can further comprise one or more nucleotides.
  • nucleotides can be, for example, deoxynucleoside triphosphates (dNTPs), for example, dTTP, dATP, dCTP, dGTP or dUTP.
  • concentration of each of the nucleotides in the composition is about 0.5 mM to about 5 mM.
  • the present compositions can further comprise one or more passive reference control.
  • the one or more passive reference control can be, for example, a ROX dye.
  • the present compositions can be formulated as concentrated stock solutions.
  • such concentrated stock solutions can be from about a 2X to about a 6X stock solution.
  • such stock solutions can be diluted for subsequent use in, for example, nucleic acid synthesis methods.
  • the compositions can be formulated as at least a 4X stock solution.
  • the present disclosure are also directed to methods for performing qPCR or RT-qPCR of a nucleic acid sample.
  • the present methods can comprise the steps of: (i) mixing a composition comprising at least one reverse transcriptase, at least one DNA polymerase, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or RT-qPCR, with: (a) a nucleic acid sample; (b) one or more labeled probes; and (c) one or more primers; and (ii) performing said qPCR or RT-qPCR on said nucleic acid sample.
  • qPCR or RT-qPCR can be performed in a single vessel (e.g., tube, compartment, well) or in a single reaction mixture.
  • compositions comprising at least one reverse transcriptase, at least one DNA polymerase, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction, are mixed with (a) a nucleic acid sample; (b) one or more labeled probes; or (c) one or more primers.
  • the one or more labeled probes can be a TaqMan® probe.
  • compositions comprising at least one qPCR inhibitor blocking agent can increase tolerance to various qPCR inhibitors (herein referred to as “inhibitor tolerance”), if present.
  • the increase in inhibitor tolerance can be indicated by a decrease in Ct.
  • Ct is decreased by at least about 10% when compared to methods using compositions that do not comprise any qPCR inhibitor blocking agents.
  • Ct can be decreased by at least 1. .
  • the composition is free or substantially free of fish gelatin.
  • the present disclosure is directed to methods for amplifying a nucleic acid molecule.
  • the methods for amplification can comprise the steps of: (i) mixing a nucleic acid template with a composition comprising one or more reverse transcriptases, one or more DNA polymerases, and one or more stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction, to form a reaction mixture; and (ii) incubating the reaction mixture under conditions sufficient to amplify a nucleic acid molecule complementary to all or a portion of said nucleic acid template.
  • the nucleic acid template can be RNA or DNA.
  • nucleic acid amplification can be performed by PCR.
  • the PCR can be qPCR.
  • the PCR can be performed by RT-qPCR.
  • the PCR can be endpoint PCR.
  • the PCR can be multiplex PCR.
  • the PCR can comprise thermal cycling. In some embodiments, the thermal cycling can be optimized for fast thermal cycling.
  • the methods for nucleic acid synthesis can further comprise incubating one or more first nucleic acid molecules under conditions sufficient to produce or synthesize one or more second nucleic acid molecules complementary to all or a portion of one or more first nucleic acid molecules.
  • reaction mixtures comprising: (a) at least one reverse transcriptase; (b) at least one polymerase; (c) at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction; and (d) at least one primer.
  • the reaction mixture can further comprise a nucleic acid template (e.g., RNA or DNA).
  • the reaction mixtures can further comprise a labeled probe (e g., TaqMan® probe).
  • the compositions can be formulated as 4X stock solutions.
  • the compositions can be used for qPCR, RT-qPCR methods, nucleic acid synthesis methods, or nucleic acid amplification (e.g., PCR) methods.
  • the qPCR or RT-qPCR methods can comprise multiplexing.
  • composition comprising a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.
  • said reverse transcriptase comprises a thermostable reverse transcriptase.
  • said DNA polymerase comprises a thermostable DNA polymerase.
  • said thermostable DNA polymerase comprises Taq DNA polymerase or a mutant, variant, or derivative thereof.
  • thermostable DNA polymerase comprises a recombinant Taq DNA polymerase or a mutant, variant, or derivative thereof.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is at least one selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is at least one selected from the group consisting of magnesium sulfate and potassium sulfate.
  • the composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol. [0046] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises a silicone-based emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • the composition further comprises a hot-start component that reversibly inactivates the at least one DNA polymerase.
  • the composition excludes gelatin or fish gelatin.
  • the composition excludes uracil-DNA glycosylase (UDG) or uracil-
  • UNG N-glycosylase
  • the composition further comprises a passive reference control.
  • said passive reference control comprises a ROX dye.
  • said composition is for use in nucleic acid synthesis, nucleic acid amplification, a quantitative polymerase chain reaction (qPCR), or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR).
  • qPCR quantitative polymerase chain reaction
  • RT-qPCR quantitative reverse transcriptase polymerase chain reaction
  • a method of performing a quantitative polymerase chain reaction (qPCR) or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) on a nucleic acid sample comprising mixing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or said RT-qPCR, with the nucleic acid sample; at least one primer pair having specificity for a nucleic acid in the nucleic acid sample; at least one labeled probe having specificity for amplicons complementary to all or a portion of the nucleic acid; and performing said qPCR or said RT-qPCR on said nucleic acid sample.
  • qPCR quantitative polymerase chain reaction
  • RT-qPCR quantitative reverse transcriptase polymerase chain reaction
  • said at least one stabilizing agent comprises an RNA aptamer that interacts with the reverse transcriptase to increase the stability of the assembled reactions.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • said labeled probe is a dual -labeled probe with a reporter dye and a quencher dye.
  • said qPCR or said RT-qPCR is performed in a single tube or a single reaction.
  • said performing said qPCR or said RT-qPCR comprises thermal cycling or fast thermal cycling.
  • said quantitative polymerase chain reaction (qPCR) or said quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is a multiplex reaction capable of detecting up to six target nucleic acids.
  • a method for amplifying a nucleic acid comprises mixing a nucleic acid template with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incubating said reaction mixture under conditions sufficient to amplify the nucleic acid, the nucleic acid being complementary to all or a portion of said nucleic acid template.
  • a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incuba
  • said nucleic acid template is a template having RNA.
  • said nucleic acid is DNA.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • a method for nucleic acid synthesis comprising mixing a sample having first nucleic acid with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or a portion of said first nucleic acid.
  • a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or
  • said first nucleic acid is RNA.
  • said second nucleic acid is DNA.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • a reaction mixture comprising a master mix composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; at least one primer having a sequence specific to a target nucleic acid in a sample mixed with said reaction mixture in the assembled polymerase chain reaction; and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • said at least one labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.
  • said target nucleic acid is RNA
  • the amplicons are DNA
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with said at least one reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the master mix composition further comprises an non-ionic silicone emulsifier.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • kits comprising a single container containing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.
  • a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.
  • said kit is used for qPCR or RT-qPCR.
  • said kit is used for nucleic acid synthesis.
  • said kit is used for nucleic amplification.
  • the kit further comprises another container containing a second composition including at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction, and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • the kit further comprises another container containing at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction; and a further container containing at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • said at least one probe is a dual-labeled probe with a reporter dye and a quencher dye.
  • said reverse transcriptase is a Moloney Murine Leukemia virus (M- MLV) reverse transcriptase or any mutant, variant or derivative thereof having reverse transcriptase activity.
  • M- MLV Moloney Murine Leukemia virus
  • said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with said reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanedioL
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • FIG. 1 depicts an one-step RT-PCR according to exemplary embodiments.
  • FIG. 4 shows amplification plots of different targets using an exemplary composition.
  • FIG. 5 is a graph showing tolerance of exemplary compositions including an inhibitor blocking agent to the inhibitor heparin.
  • FIG. 6 is a graph showing tolerance of exemplary compositions including a polyoxyethylene surfactant to the inhibitor heparin.
  • the present disclosure is directed to compositions, methods, and kits for use in the production or analysis of nucleic acids.
  • the present disclosure offers several advantages compared to known compositions or methods for the generation or amplification of nucleic acids by, for example, qPCR or RT-qPCR.
  • nucleic acid targets e.g., up to six nucleic acid targets
  • qPCR or RT-qPCR which allow for higher volume sample input if or when sample template concentrations are low (which is often the case for viral targets or forensic analysis); m) providing compositions for use in nucleic acid synthesis (e.g. qPCR or RT-qPCR) that have increased tolerance to various PCR inhibitors; n) providing compositions for use in nucleic acid synthesis (e.g.
  • nucleic acid synthesis e.g., qPCR or RT-qPCR
  • nucleic acid synthesis e.g., qPCR or RT-qPCR
  • p providing nucleic acid synthesis (e.g., qPCR or RT-qPCR) compositions and methods which allow for the increased capability to multiplex (e.g., amplify a multiplicity of targets using a single sample (e.g., two targets on one sample) or multiple samples (e.g., two targets on two different samples) in a single reaction at substantially the same time); and/or q) providing increased multiplexed nucleic acid synthesis (e.g., qPCR or RT-qPCR) compositions and methods which allow for the type of identification and quantification of nucleic acids (e.g., oncoviral nucleic acid) in a single reaction thereby reducing the amount of sample used as well as reducing costs and the
  • FIG. 1 depicts an one-step RT-PCR according to exemplary embodiments.
  • the present disclosure provides for a true “one tube/one step” procedure for qPCR, RT-PCR, or RT-qPCR.
  • the reverse transcriptase and the DNA polymerase are premixed into a single tube, allowing both the RT and the subsequent PCR step to be performed in a single reaction.
  • the advantages of one-step RT-PCR over two-step RT-PCR include fast and simple analysis, less pipetting steps, lower risk of errors and contamination, and suitability for high-throughput applications.
  • One-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube performed in a single reaction. This reaction setup can help simplify workflow, reduces variation, and minimizes possible contamination.
  • One-step RT-PCR allows easy processing of large numbers of samples, making it amenable to high-throughput applications.
  • One-step RT-PCR uses gene-specific primers for amplification, limiting the analysis to a few genes per RNA sample. Use of a gene-specific primer in RT-PCR can help maximize the yield of the target cDNA and minimize background amplification.
  • an assembled PCR reaction is a PCR reaction including all of the necessary components for performing first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube.
  • an assembled PCR reaction is a PCR reaction including all of the necessary components for performing first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction.
  • compositions comprising a variety of components in various combinations.
  • Such components can include one or more enzymes having reverse transcriptase activity, one or more DNA polymerases, one or more osmoprotectants, such as a nonionic polyoxyethylene surfactant and/or inhibitor blocking agent, or one or more stabilizing agents.
  • Additional components can also include, for example, one or more primers, one or more deoxyribonucleoside triphosphates (dATPs), one or more antibodies, one or more ribonuclease inhibitors, one or more preservative, and one or more passive reference dyes, as well as suitable PCR buffer components, including, but not limited to, glycerol and tris-buffered saline, which may include bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the composition may further include nuclease-free water.
  • Such compositions may be formulated for high-order multiplex reactions, use of RNA or DNA as a starting material, lab material or liquid handlers that require benchtop stability, and inhibitor intolerance.
  • compositions can be formulated as concentrated stock solutions (e.g., 2X, 3X, 4X, 6X, 10X, etc.) or as working solutions (e.g., IX).
  • the concentrated stock solution is at 4X.
  • having the composition as a concentrated (e.g., 4X) stock solution allows a greater amount of nucleic acid sample to be added (such as, for example, when the compositions are used for nucleic acid synthesis).
  • These compositions can be used in the present methods to produce, analyze, quantitate, and otherwise manipulate nucleic acid molecules using a one-step (or coupled) qPCR or RT-qPCR procedure.
  • the compositions of the present disclosure can be formulated as master mixes.
  • Master mixes improve the efficiency and reduce the errors associated with the assembly of large number of reactions required for high-throughput analysis.
  • master mixes can contain combination of reagents common to all reactions.
  • the master mix can contain a buffer, a salt, such as magnesium sulfate (MgSO4) or potassium sulfate (K2SO4), nucleotides, a labeled probe or dye, a reverse transcriptase, a thermostable DNA polymerase, a stabilizing agent, and/or an osmoprotectant.
  • MgSO4 magnesium sulfate
  • K2SO4 potassium sulfate
  • the present compositions can be packaged in a suitable container or vessel capable of holding the composition and which does not significantly interact with components of the composition.
  • the container or vessel can be designed to permit easy dispensing of the dosage form by individuals or by a liquid handling instrument.
  • the containers or vessels of such composition can be further packaged into multi-pack units.
  • compositions of the present disclosure comprise polypeptides having reverse transcriptase activity (i.e., reverse transcriptases).
  • the polypeptides having reverse transcriptase activity are thermostable.
  • thermostable refers to an enzyme that is heat stable or heat resistant.
  • a thermostable polypeptide having reverse transcriptase activity can also be defined as a polypeptide having reverse transcriptase activity which retains a greater percentage of its activity after a heat treatment than is retained by a polypeptide having reverse transcriptase activity that has wild-type thermostability after an identical treatment.
  • enzymes having reverse transcriptase activity can be, for example, retroviral reverse transcriptases such as M-MLV reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, Avian Sarcoma Leukosis Virus (ASLV) reverse transcriptases, as well as Lenti virus reverse transcriptases, or corresponding mutants, variants or derivatives thereof having reverse transcriptase activity.
  • retroviral reverse transcriptases such as M-MLV reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcript
  • mutants, variants, or derivatives refer to all permutations of a chemical species, which can exist or be produced, that still retains the definitive chemical activity of that chemical species.
  • Some preferred embodiments include enzymes that are RNase H+ enzymes such as, for example, RNase H+ M-MLV or RNase H+ AMV reverse transcriptases.
  • the reverse transcriptases used in the present compositions 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, pp.23-24 (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, et al., Nucl. Acids Res. 16:265-277 (1988) and in Gerard, et al., FOCUS (Life Technologies) 14:91-93 (1992), the disclosures of which are fully incorporated herein by reference in there entireties.
  • Tth reverse transcriptase which has reverse transcriptase activity in the presence of Mn 2+ and DNA polymerase activity in the presence of Mg 2+
  • Tth reverse transcriptases which has reverse transcriptase activity in the presence of Mn 2+ and DNA polymerase activity in the presence of Mg 2+
  • Methods for the isolation or purification of reverse transcriptases have been described, for example, in U.S. Pat. Nos. 4,943,531 and 5,017,492, the disclosures of which are fully incorporated herein by reference in their entireties. It is to be understood that a variety of reverse transcriptases can be used in the present disclosure, including reverse transcriptases not specifically disclosed above, without departing from the scope or preferred embodiments thereof.
  • any number of mutations can be made to the reverse transcriptases and, in a preferred embodiment, multiple mutations can be made which result in an increased reverse transcriptase stability or functionality.
  • Enzymes for use herein can also include those in which terminal deoxynucleotidyl transferase (TdT) activity has been reduced, substantially reduced, or eliminated.
  • TdT terminal deoxynucleotidyl transferase
  • 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 (the disclosures of which are fully incorporated by reference in their entireties).
  • such mutated reverse transcriptases can include reverse transcriptases with one or more alterations at amino acid positions equivalent or corresponding to Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, or F309 of M-MLV reverse transcriptase.
  • Such mutations can include point mutations, frame shift mutations, deletions and insertions, with one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.) point mutations preferred.
  • mutant or modified reverse transcriptases can be made by recombinant techniques.
  • a number of cloned reverse transcriptase genes are available or can be obtained using standard recombinant techniques (see, e.g., U.S. Pat. No. 5,668,005 and PCT Publication No. WO 98/47912).
  • oligonucleotide site-directed mutagenesis can be used to create a mutant polymerase which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule.
  • mutations can also be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor.
  • Polypeptides having reverse transcriptase activity can be added to the present compositions to give a final concentration in a working solution of about 0.001 U/pL to about 500 U/pL, about 0.005 U/pL to about 100 U/pL, about 0.01 U/pL to about 50 U/pL, about 0.05 U/pL to about 20 U/pL, about 0.1 U/pL to about 10 U/pL, about 0.2 U/pL to about 5 U/pL, or preferably at a concentration of about 0.2 U/pL, about 0.4 U/pL, about 0.8 U/pL, about 1.0 U/pL, about 1.2 U/pL, about 1.4 U/pL, about 1.8 U/pL, about 2 U/pL, about 3 U/pL, about 4 U/pL, or about 5 U/pL.
  • compositions of the present disclosure can also comprise one or more polymerases.
  • polymerases can be any enzyme capable of replicating a DNA molecule.
  • the DNA polymerases are thermostable DNA polymerases.
  • 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 doublestranded nucleic acids during PCR amplification. Irreversible denaturation of the enzyme refers to substantial loss of enzyme activity.
  • a thermostable DNA polymerase will not irreversibly denature at about 90 °C- 100 °C under conditions such as is typically required for PCR amplification.
  • DNA polymerases in accordance with the present disclosure 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).
  • thermostable polymerases Suitable for use as sources of thermostable polymerases or the genes thereof for expression in recombinant systems are, for example, the thermophilic bacteria Thermus thermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosii 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.
  • DNA polymerases from other organisms can also be used herein without departing from the scope or preferred embodiments thereof.
  • DNA polymerases 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).
  • thermostable DNA polymerases for use in the present compositions and methods include, but are not limited to, Taq, Tne, Tma, Tfi/VENT, DEEPVENT, Pfu, Pwo, Tfi or Tth DNA polymerases, or mutants, variants or derivatives thereof having DNA polymerase activity.
  • 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, the thermophilic bacterium Thermus aquaticus, 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).
  • thermostable DNA polymerase is AmpliTaqTM Fast DNA Polymerase (Roche Molecular Systems, Inc., Pleasanton, CA) or AmpliTaqTM Gold Fast PCR (Applied Biosystems).
  • 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.
  • DNA polymerases can be used in the present compositions, methods and kits, including polymerases not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.
  • thermophilic DNA polymerases particularly of Tne and Tma polymerases are disclosed, for example, in U.S. Application Nos. 08/689,807 and 08/689,818, both filed Sept. 6, 1996, both of which are incorporated by reference herein in their entireties.
  • Tfi, Tli/VENT, and DEEPVENT are available commercially (e.g., from New England BioLabs; Beverly, MA), or can be produced as described (Bej and Mahbubani, in: PCR Technology: Current Innovations, Griffin, H. G., and Griffin, A. M., eds., CRC Press, pp.
  • Thermostable DNA polymerases of the present disclosure can be added to the present compositions to give a final concentration in a working solution of 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, about 0.005 U/pL to about 2 U/pL, about 0.01 U/pL to about 1 U/pL, about 0.02 U/pL to about 0.5 U/pL, or preferably at a concentration of about 0.02 U/pL, about 0.04 U/pL, about 0.08 U/pL, about 0.1 U/pL, about 0.12 U/pL, about 0.14 U/pL, about 0.18 U/pL, about 0.2 U/pL, about 0.3 U/pL, about
  • the concentration of DNA polymerases can be determined as a ratio of the concentration of the enzymes having reverse transcriptase activity to the concentration of the enzymes having DNA polymerase activity.
  • the unit ratio of the reverse transcriptase enzymes to the DNA polymerase enzymes can range from about 500 U/pL to about 0.001 U/pL, about 250 U/pL to about 0.005 U/pL, about 100 U/pL to about 0.01 U/pL, about 50 U/pL to about 0.05 U/pL, about 25 U/pL to about 0.1 U/pL, or preferably about 5 U/pL to about 0.5 U/pL.
  • suitable ratios of unit activities of reverse transcriptase enzymes to DNA polymerases suitable for use in the present compositions, methods and kits will be apparent to one of ordinary skill in the art.
  • one or more stabilizing agents can be included in the present compositions.
  • the stabilizing agent increases the stability of assembled reactions during a PCR process.
  • the stabilizing agent includes an RNA aptamer.
  • the stabilizing agent increases the reaction efficiency during a PCR process, especially when more than one target is present in the sample.
  • the stabilizing agent permits an increased level of the DNA polymerase to allow balancing of benchtop stability with multiplex capacity.
  • one or more qPCR inhibitor blocking agents and/or osmoprotectants can be added to the present compositions to assist in overcoming the inhibition of PCR reactions by a variety of compounds often found in samples used for nucleic acid preparation, isolation or purification.
  • 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).
  • compositions can further comprise agents that work alone or in combination to increase tolerance to various qPCR inhibitors including, for example, humic acid, hematin, and heparin.
  • Such qPCR inhibitor blocking agents for use in the present disclosure can include nonionic surfactants such as, but not limited to, nonionic polyoxyethylene surfactants, such as polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate, or neutral compounds with a cationic group lacking a hydrogen, such as, but not limited to, ethylene glycol, betaine and 1,2-propanediol.
  • nonionic surfactants such as, but not limited to, nonionic polyoxyethylene surfactants, such as polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate, or neutral compounds with a cationic group lacking a hydrogen, such as, but not limited to, ethylene glycol, betaine and 1,2-propanediol
  • a silicone-based emulsifier such as antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15 may be included to counter any foaming tendency of the nonionic surfactant.
  • a Ct value can be determined using a derivative of a PCR curve.
  • a first, second, or nth order derivative method can be performed on a PCR curve in order to determine a Ct value.
  • a characteristic of a derivative can be used in the determination of a Ct value. Such characteristics can include, but are not limited to, 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.
  • a Ct value can be determined using a thresholding and baselining method.
  • an upper boundary 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 boundary to an exponential phase 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 include, but are not limited to, various embodiments of a fit point method, and various embodiments 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 Publication No. 2004/0096819; the disclosures of which are herein incorporated by reference in their entireties).
  • qPCR inhibitor blocking compounds or agents can be added to the present compositions to give a final concentration in a working solution of about 1 ng/pL to about 10,000 ng/pL, about 50 ng/pL to about 8000 ng/pL, about 100 ng/pL to about 6000 ng/pL, about 200 ng/pL to about 3000 ng/pL or preferably about 500 ng/pL to about 1000 ng/pL.
  • qPCR inhibitor blocking agents can also be added as a percentage of the final concentration in a working solution, for example, from about 0.001% to about 15%, about 0.05% to about 10%, about 0.01% to about 5%, or preferably about 0.1% to about 1%.
  • qPCR inhibitor blocking agents can reduce the amount of qPCR inhibition by such qPCR inhibitors by at least 1 to 100% compared to the level of inhibition observed in the absence of such qPCR inhibitor blocking agents.
  • inhibition can be reduced by at least about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100% or any percentage in between.
  • compositions of the present disclosure can further comprise one or more nucleotides (e.g., deoxynucleoside triphosphates (dNTPs)).
  • dNTPs deoxynucleoside triphosphates
  • the nucleotide components of the present compositions serve as the “building blocks” for newly synthesized nucleic acids, being incorporated therein by the action of the reverse transcriptases or DNA polymerases.
  • nucleotides suitable for use in the present compositions include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, diTP, 7-deaza-dGTP, a-thio-dATP, a-thio-dTTP, a-thio-dGTP, a-thio- dCTP or derivatives thereof, all of which are available commercially from sources including Life Technologies (Carlsbad, CA), New England BioLabs (Beverly, MA) and Sigma Chemical Company (Saint Louis, MO).
  • dNTPs may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g., 3 H, 14 C, 32 P or 35 S), vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin (DIG) and the like. Labeled dNTPs may also be obtained commercially, for example from Life Technologies (Carlsbad, CA) or Sigma Chemical Company (Saint Louis, MO).
  • radioisotopes e.g., 3 H, 14 C, 32 P or 35 S
  • vitamins e.g., biotin
  • fluorescent moieties e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin
  • chemiluminescent labels chemiluminescent labels
  • DIG dioxigen
  • fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TA MRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRl 10]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Life Technologies, Foster City, CA.
  • DIG labels include digoxigenin-11-UTP available from Boehringer Mannheim, Indianapolis, IN, and biotin labels include biotin-21-UTP and amino-7-dUTP available from Clontech, Palo Alto, CA.
  • nucleotide includes modified nucleotides. Many examples of modified nucleotides are disclosed in U.S. Patent No. 6,200,757 (the disclosure of which is herein incorporated by reference in its entirety).
  • dNTPs can be added to give a final concentration in a working solution of each dNTP 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 0.6 mM.
  • the present compositions can comprise one or more primers which facilitate the synthesis of a first DNA molecule complementary to all or a portion of an RNA template (e.g., a single-stranded cDNA molecule). Such primers can also be used to synthesize a DNA molecule complementary to all or a portion of the first DNA molecule, thereby forming a double-stranded cDNA molecule. 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, 10, 20, etc.) nucleotides in length.
  • primers include, but are not limited to, target-specific primers (which are preferably gene-specific 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 can be useful in the present compositions, methods and kits, including those not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.
  • the final concentration of primers in a working solution can range from about 25 nM to about 2000 nM, such as about 50 nM to about 1700 nM, about 75 nM to about 1500 nM, about 100 nM to about 1200 nM, about 200 nM to about 1000 nM, or any range in between.
  • the concentration of the primers is between about 400 nM to about 900 nM.
  • compositions can further comprise probes for the detection of target nucleic acids.
  • probes are known in the art, for example, TaqMan® probes (see, e.g., U.S. Patent No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Patent Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology 14:303-308 (1996)), stemless or linear beacons (see, e.g., PCT Publication No. WO 99/21881), PNA Molecular BeaconsTM (see, e.g., U.S. Patent Nos.
  • Probes can comprise reporter dyes such as, for example, 6-carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET) and the like.
  • Detector probes can also comprise quencher moieties such as tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch Technologies, Novato, CA), Iowa Black (IDT, Coralville, IA), QSY quencher (Molecular Probes, Eugene, OR), and DABSYL and DABCEL sulfonate/carboxylate Quenchers (Epoch Biosciences, Bothell, WA) and the like.
  • TAMRA tetramethylrhodamine
  • TAMRA Black Hole Quenchers
  • ITT Iowa Black
  • QSY quencher Molecular Probes, Eugene, OR
  • DABSYL and DABCEL sulfonate/carboxylate Quenchers Epoch Biosciences, Bothell,
  • Probes can also comprise 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.
  • Exemplary detectable labels include, for instance, 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.
  • 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
  • Suitable detectable labels may 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, Fl-Ceramide, R6G SE, T
  • EGFP blue fluorescent protein
  • BFP blue fluorescent protein
  • 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/BODIPY® FL, Fluorescein/QSY7 and QSY9
  • 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, LysoSen
  • 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.
  • 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 compositions, methods and kits, including those not specifically disclosed herein.
  • the final probe concentration in a working solution 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 some exemplary embodiments, the probe concentration is between about 100 nM to about 250 nM.
  • additives capable of facilitating or enhancing reverse transcription, amplification, or a combination of both reactions are known in the art.
  • agents for facilitating or enhancing RT-qPCR are known in the art.
  • one or more of these additives can be incorporated in the present compositions to optimize the generation and replication of nucleic acids from a ribonucleic acid or deoxyribonucleic acid templates.
  • Additives can be organic or inorganic compounds.
  • Some additives useful in the present compositions, methods and kits include polypeptides as well as nonpolypeptide additives. Such additives can include, for example, RNase inhibitor protein (RIP), lectins, E.
  • RIP RNase inhibitor protein
  • lectins E.
  • coli single- stranded binding (SSB) protein tRNA, rRNA, 7-deaza-2'-deoxyguanosine (dC7GTP), sulfur-containing compounds, acetate-containing compounds, dimethylsulfoxide (DMSO), glycerol, formamide, betaine, tetramethylammonium chloride (TMAC), polyethylene glycol (PEG), various surfactants or generally any zwitterionic, cationic, anionic or non-ionic (e g., TWEEN 20, NP-40, Triton X-100, and CHAPS) detergents, ectoine, sodium azide, kathon, and polyols, to name just a few.
  • compositions and methods in accordance with the present disclosure can also include additional “hot start” PCR components or steps, as a means to further prevent, reduce or eliminate nonspecific nucleic acid synthesis.
  • hot start refers to any modified form of PCR which prevents non-specific amplification of DNA by inactivating the polymerase activity at lower annealing temperatures and reactivating or activating the polymerase activity at higher temperatures during the extension phase.
  • hot start mechanisms are well known to those of ordinary skill in the art and will be readily selectable based on their ability to work in accordance with the present disclosure.
  • the hot start components that can be optionally added to the present compositions can include, for example, an antibody or antibodies, specially designed primers, competitive oligonucleotides or aptamers, polymerase binding proteins or sequestration beads.
  • Sequestration wax beads for hot start PCR are commercially available, e g., AmpliWax® PCR Gem 100 and AmpliWax® PCR Gem 50 (Applied Biosystems, Foster City, CA). Selection of a suitable aptamer can be performed by a method known in the art or a commercially available aptamer can be used. Similarly, selection of a suitable primer can be performed by a method known in the art or a commercially available primer can be used. In some cases, a suitable primer can be a primer specially designed to have secondary structures that prevent the primers from annealing until cycling temperatures denature them. Antibodies for hot start PCR can be generated or selected by a method known in the art.
  • a commercially available antibody can be used, for example, the TaqStart® Antibody (Clontech, Mountain View, CA) which is effective with any Taq-derived DNA polymerase, including native, recombinant, and N-terminal deletion mutants.
  • An appropriate concentration of the reagent for hot start PCR in the assembled PCR can be determined by a method known in the art or, for a commercial product, as suggested by the manufacturer.
  • Other examples of hot start components or mechanisms used for this purpose are known in the art (see, e.g., U.S. Patent No. 6,403,341 and U.S. Patent Application Publication No. 2009/0269766, the disclosures of which are fully incorporated herein by reference in their entireties.)
  • compositions and methods in accordance with the present disclosure can also include a passive reference control.
  • the passive reference control is used to minimize sample-to-sample or well-to-well variations in quantitative real-time nucleic aciddetection assays and can be included at a concentration allowing its use as detectable control.
  • a reference chromophore specifically a fluorophore, is included as the passive reference control.
  • the reference chromophore is the dye ROX (Invitrogen, Carlsbad, CA).
  • ROX can be included in the composition at a concentration in a working solution of about 40 nM to about 80 nM, specifically about 60 nM.
  • one or more reverse transcriptases and one or more DNA polymerases are preferably mixed in a buffered salt solution.
  • buffer agents or salt solutions used in the present compositions and reaction mixtures provide appropriate pH and ionic conditions to maintain stability of the enzymes having reverse transcriptase activity or DNA polymerase activity.
  • stable and “stability” 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 week 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.
  • 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 week at a temperature of about 4 °C, about two to six months at a temperature
  • buffering agents can include, for example, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS.
  • the buffering agent includes tris with BSA to provide a buffered pH of about 8.
  • salt solutions can include, for example, 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. It is to be understood that a wide variety of buffers and salt solutions are known in the art that can be used in the present compositions, methods and kits, including those not specifically disclosed herein.
  • the compositions can be provided as a concentrated stock.
  • concentration 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 reverse transcription of nucleic acids).
  • working solution can be used to refer to the solution having an optimal concentration to perform a particular function.
  • compositions of the present disclosure can be stock solutions of about 2X, about 3X, about 4X, about 5X, about 6X, about 10X, and so on. In some preferred embodiments, the stock solution is about 4X.
  • compositions can require greater than 2X, greater than 3X, greater than 4X, greater than 5X, greater than 6X, greater than 10X, and so on, dilution to be at working, or optimal, concentration for use in nucleic acid synthesis methods.
  • Nucleic acid samples suitable for use in accordance with the present disclosure can include any quantity of one or more nucleic acid molecules.
  • such nucleic acid molecules can be extracellular nucleic acid molecules.
  • such nucleic acid molecules can be derived from cells.
  • such cells can include, for example, any prokaryotic, eukaryotic or plant cell.
  • Such cells can be normal cells, diseased cells, transformed cells, established cells, progenitor cells, precursor cells, fetal cells, embryonic cells, bacterial cells, yeast cells, animal cells (including human cells), avian cells, plant cells and the like, or tissue isolated from a plant or an animal (e.g., human, cow, pig, mouse, sheep, horse, monkey, canine, feline, rat, rabbit, bird, fish, insect, etc.).
  • nucleic acid molecules can also be isolated from viruses.
  • such nucleic acid samples can be extracted from a variety of sources. These include, but are not limited to, for example clothing, soil, skin, hair, blood, serum, feces, milk, saliva, urine, or other secretory fluids. These sources can also contain compounds that inhibit PCR amplification.
  • the nucleic acid samples or templates can be any ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) of interest, known or unknown, to the practitioner.
  • Nucleic acid samples can be artificially synthesized or isolated from natural sources.
  • the nucleic acid sample is single stranded.
  • the nucleic acid sample can be double stranded.
  • the nucleic acid sample can be messenger RNA (mRNA), RNA, genomic DNA (gDNA) or cDNA. Many nucleic acid sample preparation or isolation methods are known in the art.
  • nucleic acid isolation or preparation kits are also available commercially, for example, MagMAXTM (Applied Biosystems, Foster City, CA), iPrepTM (Invitrogen, Carlsbad, CA) and QIAmp MinElute (Qiagen, San Diego, CA).
  • such methods can comprise incubating the mixture comprising one or more reverse transcriptases or one or more polymerases under conditions sufficient to make a nucleic acid molecule or molecules complementary to all or a portion of the one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.) templates.
  • a primer e.g., an oligo(dT) primer
  • one or more nucleotides are preferably used for nucleic acid synthesis in the 5' to 3' direction.
  • Additional embodiments provide methods for amplifying a nucleic acid molecule comprising contacting the nucleic acid molecule with a polymerase.
  • simplex (i.e., single) amplification reactions are performed at one time in a single reaction or vessel.
  • multiplex (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 and so on) amplification reactions are performed at one time in a single reaction or vessel.
  • multiplex or “multiplexing” refers to the essentially simultaneous amplification or analysis of multiple targets in a single reaction.
  • multiplexing can involve the amplification of a single or multiple targets from one or multiple sample input(s) and an exogenous control target from one exogenous control template within the same reaction vessel (e.g., tube, compartment, well).
  • such methods can comprise one or more polymerase chain reactions (PCRs).
  • PCRs polymerase chain reactions
  • multiplex PCR reactions can comprise the essentially simultaneous amplification of greater than 1, greater than 2, greater than 3, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 20, greater than 50, greater than 100, greater than 1000, etc. nucleic acid targets within the same reaction.
  • such PCR methods can be quantitative PCR (qPCR) or end point PCR amplification methods.
  • such PCR methods are real time PCR amplification methods.
  • thermal cycling can comprise alternating heating and cooling of the mixture sufficient to amplify the DNA molecule and which most preferably comprises alternating from a first temperature range of from about 90 °C to about 100 °C, to a second temperature range of from about 45 °C to about 75 °C, from about 50 °C to about 70 °C, from about 55 °C to about 65 °C, or preferably at about 58 °C, at about 59 °C, at about 60 °C, at about 61 °C or at about 62 °C.
  • the thermal cycling can be performed any number of times, such as any number greater than about 10 times, greater than about 20 times, greater than about 30 times, or from about 5 to about 80 times, about 10 to about 70 times, about 20 to about 60 times, or preferably from about 30 to about 50 times.
  • the thermal cycling can be optimized for fast thermal cycling.
  • Such protocols and apparatuses for fast thermal cycling can be found in, for example, U.S. Patent Nos. 6,210,882, or Kopp, et al., Science 280: 1046-1048 (1998); Chiou, et al., Anal. Chem. 73:2018-2021 (2001) (for modified electric heating elements); Kalinina, et al., Nucleic Acids Res.
  • Such PCR thermal cycling can be performed on a variety of instruments known to those of skill in the art.
  • Some instruments can be commercially available, for example, from Applied Biosystems (e.g., AB SDS Instruments 7300 Real-Time PCR System, 7500 Real-Time PCR System, 7500 Fast Real-Time PCR System, 7900HT Real-Time PCR System, StepOne Real-Time PCR System and StepOne Plus Real-Time PCR System, or ViiA 7 Real-Time PCR System). It is to be understood that a wide variety of instruments are known in the art that may be useful in the present methods, including those not specifically disclosed herein.
  • the present compositions can be used in methods for one-step (or coupled) RT-qPCR.
  • RT-qPCR reaction mixtures are incubated at a temperature sufficient to synthesize a DNA molecule complementary to all or portion of an RNA template (e.g., cDNA) and then incubated at a second temperature sufficient to amplify newly synthesized cDNA molecules.
  • temperatures used for cDNA synthesis can range from about 30 °C to about 75 °C, about 35 °C to about 70 °C, about 40 °C to about 60 °C, or preferably from about 45 °C to about 55 °C.
  • temperatures used for cDNA amplification can range from about 40 °C to about 80 °C, about 45 °C to about 75 °C, about 50 °C to about 70 °C or preferably from about 55 °C to about 65 °C.
  • the use of at least one stabilizing agent can increases the reaction efficiency during a PCR process, especially when more than one target is present in the sample.
  • the use of at least one stabilizing agent permits an increased level of the DNA polymerase to allow balancing of benchtop stability with multiplex capacity.
  • the use of at least one qPCR inhibitor blocking agent can increase tolerance to one or more qPCR inhibitors.
  • increased tolerance can be indicated by, for example, a decrease in Ct or increase in dRn (e.g., when analyzed by real time PCR) or by an increase in the amount of amplified product (e.g., when analyzed by agarose gel electrophoresis).
  • qPCR inhibitor tolerance (as determined by Ct) can be increased by at least about 10% (e.g., about 10%, about 20%, about 30%, about 40%, about 60%, about 80%, etc.) when using at least one qPCR inhibitor blocking agent compared to methods that do not.
  • Ct value is decreased at least one (e.g., at least 1, at least 2, at least 3 at least 5, at least 10, etc.) compared to the Ct value achieved for methods that employ compositions without qPCR inhibitor blocking agents.
  • compositions comprising at least 500 ng/pL BSA can decrease Ct by at least 8 for reactions comprising at least 40 pM hematin, or by at least 7 for reactions comprising at least 10 ng/pL humic acid.
  • methods utilize compositions that are free or substantially free of gelatins, including fish gelatin.
  • a neutral compound with a cationic group lacking a hydrogen such as, for example, ethylene glycol, betaine and 1,2-propanediol, serves as a qPCR inhibitor blocking agent.
  • a nonionic polyoxyethylene surfactant such as, for example, Brij 35, also serves as a qPCR inhibitor blocking agent.
  • an RNA sample is stored at -80 °C
  • a DNA sample is stored at -20 °C
  • a master mix composition of the present disclosure is stored at -25 °C to -15 °C.
  • an RT-qPCR mix is prepared by first thawing a sample on ice, thawing assays on ice or on the benchtop, and thawing a master mix composition of the present disclosure on ice or on the benchtop.
  • the assays are vortexed briefly to mix then centrifuged to collect.
  • Thawed samples are mixed by gentle inversion or by flicking three to five times and then centrifuged to collect the contents at the bottom of the tube. These mixing and centrifuging steps may be repeated if the compositions are not sufficiently thawed.
  • the total volume for each reaction component is then calculated according to Table 1. Working on ice, the components are added directly to each well of an optical reaction plate.
  • the components may be mixed together without the sample then added to the plate.
  • the sample may be added directly to the wells of the plate.
  • the reaction plate is covered with an optical adhesive cover and inverted three to five times, making sure that the contents of the wells are moving back and forth between the seal and the bottom of the well to ensure proper mixing.
  • the reaction plate is then centrifuged at 150 * g (1000 rpm) for one minute to collect the contents at the bottom of the wells and eliminate air bubbles.
  • Table 1 Component Amounts includes 10% overage.
  • Potential assays include the TaqManTM Assay Mix, FAMTM dye; TaqManTM Assay Mix, VICTM dye; TaqManTM Assay Mix, ABYTM dye; and TaqManTM Assay Mix, JUNTM dye, Cy®5 dye, Cy®5.5 dye, and AlexaTM647 dye. Assays can be at a concentration other than 20X. Scale the volume appropriately.
  • setting up and running the qPCR instrument includes setting up the following parameters in the qPCR system software: 20 pL recommended sample volume (may be varied), default auto increment settings value, default data collection value, and default ramp rate settings value.
  • the thermal protocol is set up according to Table 2.
  • the passive reference dye is selected, preferably between TaqPathTMDuraPlexTM 1-Step RT-qPCR Master Mix and TaqPathTMDuraPlexTM 1-Step RT-qPCR Master Mix (No ROXTM).
  • the reaction plate is then loaded into the qPCR system and the run is started.
  • Table 2 Thermal Protocol hl RT enzyme functions best in the range of 48 to 55°C.
  • Annealing temperature should be consistent with melting temperature (T m ) of primer designs.
  • the results of the run are analyzed as appropriate for the assay and instrument.
  • the standard curve method or the relative quantification method is preferably used to analyze the results.
  • the qPCR system software can be used to set the baseline and threshold values for the amplification plot. They can be set automatically or manually.
  • the baseline is the initial cycle in which there is a change in the fluorescence signal.
  • the intersection of the threshold and the amplification plot defines the Ct value.
  • the threshold is set above the background signal and within the exponential growth phase of the amplification curve.
  • General guidelines for analysis include viewing the amplification plot and then, if needed, adjusting the baseline and threshold values and reviewing the replicates and outliers.
  • the Ct (C q ) values are viewed for each well and for each replicate group.
  • the slope, amplification efficiency, R 2 value, y- intercept, Ct values, and outliers are viewed.
  • kits for use in reverse transcription or amplification of a nucleic acid molecule.
  • Kits according to this embodiment can comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, plates, bottles and the like, wherein a first container means contains one or more polypeptides of the present disclosure having reverse transcriptase activity and one or more DNA polymerases.
  • a carrier means such as a box, carton, tube or the like
  • container means such as vials, tubes, ampoules, plates, bottles and the like
  • a first container means contains one or more polypeptides of the present disclosure having reverse transcriptase activity and one or more DNA polymerases.
  • a first container means contains one or more polypeptides of the present disclosure having reverse transcriptase activity and one or more DNA polymerases.
  • they can be in a single container as mixtures of two or more (e.g., 2, 3, 4,
  • kits provided herein can also comprise (in the same or separate containers), a suitable buffer, one or more nucleotides, one or more stabilizing agents, one or more qPCR inhibitor blocking agents, one or more probes, or one or more primers.
  • a suitable buffer In some preferable embodiments, the reverse transcriptase(s), DNA polymerase(s), stabilizing agent(s), qPCR inhibitor blocking agent(s), nucleotides, and a suitable buffer are combined in a single tube or container.
  • the reverse transcription and amplification kits can comprise one or more components (in mixtures or separately) including one or more polypeptides having reverse transcriptase activity and one or more DNA polymerases.
  • Such reverse transcription and amplification kits can further comprise one or more nucleotides needed for synthesis of a nucleic acid molecule, one or more probes or one or more primers (e.g., oligo(dT) for reverse transcription).
  • Preferred polypeptides having reverse transcriptase activity, DNA polymerases, nucleotides, probes, primers and other components suitable for use in the reverse transcription and amplification kits provided herein include those described above.
  • kits encompassed by this embodiment can further comprise additional reagents and compounds necessary for carrying out standard nucleic acid reverse transcription and/or amplification protocols.
  • additional reagents and compounds necessary for carrying out standard nucleic acid reverse transcription and/or amplification protocols can be contained in one or more containers, and can be contained in such containers in a mixture of two or more of the above-noted components or can be contained in the present kits in separate containers.
  • Those of skill in the art will understand that other components, either in the same tube or in separate tubes, may also be included in the kit to further facilitate or enhance reverse transcription or amplification.
  • Such components or additives can include for example, Mg 2+ , uracil DNA glycosylase, a passive reference control to minimize sample-to-sample or well-to-well variations in quantitative real-time DNA-detection assays (e.g., dyes such as ROX) and various hot start components (e.g., antibodies, oligonucleotides, beads, etc).
  • Mg 2+ Mg 2+
  • uracil DNA glycosylase e.g., uracil DNA glycosylase
  • a passive reference control to minimize sample-to-sample or well-to-well variations in quantitative real-time DNA-detection assays
  • various hot start components e.g., antibodies, oligonucleotides, beads, etc.
  • the present kits can comprise compositions for use in nucleic acid synthesis (e.g., qPCR or RT-qPCR).
  • compositions for use in nucleic acid synthesis e.g., qPCR or RT-qPCR.
  • Such compositions can be formulated as concentrated stock solutions (e.g., 2X, 3X, 4X, 5X, 6X, etc).
  • the compositions can be formulated as concentrated stock solutions in a single tube or container, comprising one or more polypeptides having reverse transcriptase activity and one or more DNA polymerases.
  • such concentrated stock compositions can further comprise one or more stabilizing agents, one more qPCR inhibitor blocking agents, one or more nucleotides, one or more host start components, one or more passive reference controls, or one or more RNase inhibitor proteins (RIP) in a buffered solution.
  • buffer solutions can comprise glycerol, DMSO, Mg 2+ , or a detergent (such as TWEEN 20 or NP-40).
  • compositions, kits, or master mixes included in the kits described herein can be stored at about -16 °C, about -18 °C, about -20 °C, about -22 °C, about -24 °C, about -26 °C, about -28 °C, about -30 °C without freezing.
  • the compositions of the kits are stored at about -20 °C without freezing.
  • the present composition can be stored at freezing temperatures (e.g., below 0 °C, below -5 °C, below -20 °C, below, -30 °C, below -40 °C, etc.) without having to be thawed prior to use.
  • freezing temperatures e.g., below 0 °C, below -5 °C, below -20 °C, below, -30 °C, below -40 °C, etc.
  • thaw thawing or “thawed” refers to the process whereby heat changes something from a solid (e.g., frozen) to a gel or liquid.
  • the present compositions can be a liquid or a gel (or viscous liquid) at freezing temperatures.
  • such compositions, especially if in gel form can be incubated at about 4 °C prior to subsequent use to ensure proper mixing, but do not require thawing per se.
  • FIGS. 2A and 2B a PCR assay was run on a QuantStudio Flex with a 384- well block using 10-pl reaction mixtures including bacteriophage MS2 as a target and ABY as a reporter dye.
  • Cl shows amplification of amplification products prepared using an exemplary composition including a RT aptamer, a stabilizing agent and qPCR inhibitor blocking agents.
  • C2 shows amplification of amplification products prepared using a conventional composition that does not include a RT aptamer, the stabilizing agent or qPCR inhibitor blocking agents.
  • the amplification of the amplification products prepared using an exemplary composition, Cl is significantly greater after 24 hours than that of the amplification products prepared using a conventional composition, C2.
  • the exemplary composition, Cl demonstrated enhanced benchtop stability compared to the conventional composition, C2.
  • FIGS. 3 A and 3B a PCR assay was run on a QuantStudio Flex with a 384- well block using 10-pl reaction mixtures including MOB kinase activator IB as a target and FAM as a reporter dye.
  • Cl shows amplification of amplification products prepared using an exemplary composition including a RT aptamer, a stabilizing agent and qPCR inhibitor blocking agents.
  • C2 shows amplification of amplification products prepared using a conventional composition that does not include a RT aptamer, the stabilizing agent or qPCR inhibitor blocking agents.
  • the amplification of the amplification products prepared using an exemplary composition, Cl is greater after 24 hours than that of the amplification products prepared using a conventional composition, C2.
  • the exemplary composition, Cl demonstrated enhanced benchtop stability compared to the conventional composition, C2.
  • FIG. 4 shows amplification plots of different targets using an exemplary composition.
  • the exemplary composition has a wide dynamic range and high sensitivity for targets at low copy numbers in a 6-plex performed in a single well.
  • FIG. 5 is a graph showing tolerance of exemplary compositions including an inhibitor blocking agent to the inhibitor heparin.
  • FIG. 6 is a graph showing tolerance of exemplary compositions including a polyoxyethylene surfactant to the inhibitor heparin.
  • a composition comprises a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.
  • said reverse transcriptase comprises a thermostable reverse transcriptase.
  • thermostable reverse transcriptase comprises an Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or a mutant, variant, or derivative thereof.
  • M-MLV Moloney Murine Leukemia virus
  • said M-MLV reverse transcriptase comprises one or more mutations selected from the group consisting of: Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, and F309.
  • said DNA polymerase comprises a thermostable DNA polymerase.
  • said thermostable DNA polymerase comprises Taq DNA polymerase or a mutant, variant, or derivative thereof.
  • thermostable DNA polymerase comprises a recombinant Taq DNA polymerase or a mutant, variant, or derivative thereof.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is at least one selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is at least one selected from the group consisting of magnesium sulfate and potassium sulfate.
  • the composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises a silicone-based emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • the composition further comprises a hot-start component that reversibly inactivates the at least one DNA polymerase.
  • the composition excludes gelatin or fish gelatin. [0237] In an embodiment of the first aspect, the composition excludes uracil-DNA glycosylase (UDG) or uracil-N-glycosylase (UNG).
  • UDG uracil-DNA glycosylase
  • UNG uracil-N-glycosylase
  • the composition further comprises a passive reference control.
  • said passive reference control comprises a ROX dye.
  • said composition is for use in nucleic acid synthesis, nucleic acid amplification, a quantitative polymerase chain reaction (qPCR), or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR).
  • qPCR quantitative polymerase chain reaction
  • RT-qPCR quantitative reverse transcriptase polymerase chain reaction
  • a method of performing a quantitative polymerase chain reaction (qPCR) or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) on a nucleic acid sample comprises mixing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or said RT-qPCR, with the nucleic acid sample; at least one primer pair having specificity for a nucleic acid in the nucleic acid sample; at least one labeled probe having specificity for amplicons complementary to all or a portion of the nucleic acid; and performing said qPCR or said RT-qPCR on said nucleic acid sample.
  • qPCR quantitative polymerase chain reaction
  • RT-qPCR quantitative reverse transcriptase polymerase chain reaction
  • said at least one stabilizing agent comprises an RNA aptamer that interacts with the reverse transcriptase to increase the stability of the assembled reactions.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • said labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.
  • said qPCR or said RT-qPCR is performed in a single tube or a single reaction.
  • said performing said qPCR or said RT-qPCR comprises thermal cycling or fast thermal cycling.
  • said quantitative polymerase chain reaction (qPCR) or said quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is a multiplex reaction capable of detecting up to six target nucleic acids.
  • a method for amplifying a nucleic acid comprises mixing a nucleic acid template with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incubating said reaction mixture under conditions sufficient to amplify the nucleic acid, the nucleic acid being complementary to all or a portion of said nucleic acid template.
  • a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incuba
  • said nucleic acid template is a template having RNA.
  • said nucleic acid is DNA.
  • said at least one stabilizing agent comprises an
  • RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • a method for nucleic acid synthesis comprises mixing a sample having first nucleic acid with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or a portion of said first nucleic acid.
  • said first nucleic acid is RNA.
  • said second nucleic acid is DNA.
  • said at least one stabilizing agent comprises an
  • RNA aptamer interacts with the reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • a reaction mixture comprises a master mix composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; at least one primer having a sequence specific to a target nucleic acid in a sample mixed with said reaction mixture in the assembled polymerase chain reaction; and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • said at least one labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.
  • said target nucleic acid is RNA, and the amplicons are DNA.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with said at least one reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the master mix composition further comprises an non-ionic silicone emulsifier.
  • said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • a kit comprises a single container containing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.
  • said kit is used for qPCR or RT-qPCR.
  • said kit is used for nucleic acid synthesis. [0301] In an embodiment of the sixth aspect, said kit is used for nucleic amplification.
  • the kit further comprises another container containing a second composition including at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction, and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • the kit further comprises another container containing at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction; and a further container containing at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.
  • said at least one probe is a dual-labeled probe with a reporter dye and a quencher dye.
  • said reverse transcriptase is a Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or any mutant, variant or derivative thereof having reverse transcriptase activity.
  • M-MLV Moloney Murine Leukemia virus
  • said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.
  • said at least one stabilizing agent comprises an RNA aptamer.
  • said RNA aptamer interacts with said reverse transcriptase.
  • said at least one stabilizing agent further comprises a sulfate additive.
  • said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.
  • said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.
  • said composition further comprises at least one qPCR inhibitor blocking agent.
  • said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.
  • said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.
  • said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.
  • the composition further comprises an non-ionic silicone emulsifier.
  • the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.
  • compositions disclosed herein can be provided in individual containers or in a single container, as appropriate. Instructions and protocols for using the kit advantageously can be provided.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente divulgation concerne des compositions, des méthodes et des kits pouvant être employés dans la synthèse de molécules d'acides nucléiques. Plus spécifiquement, l'invention concerne des compositions, des procédés et des kits pour l'amplification de molécules d'acide nucléique dans une procédure qPCR ou RT-qPCR en une étape comprenant une transcriptase inverse, une ADN polymérase, au moins un dNTP, et au moins un agent stabilisant, ledit au moins un agent stabilisant augmentant la stabilité d'une réaction en chaîne par polymérase assemblée.
PCT/US2024/036306 2023-06-30 2024-06-30 Compositions, méthodes et kits de synthèse et d'amplification d'acides nucléiques Ceased WO2025007089A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363524605P 2023-06-30 2023-06-30
US63/524,605 2023-06-30

Publications (1)

Publication Number Publication Date
WO2025007089A1 true WO2025007089A1 (fr) 2025-01-02

Family

ID=92108413

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/036306 Ceased WO2025007089A1 (fr) 2023-06-30 2024-06-30 Compositions, méthodes et kits de synthèse et d'amplification d'acides nucléiques

Country Status (1)

Country Link
WO (1) WO2025007089A1 (fr)

Citations (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
US4943531A (en) 1985-05-06 1990-07-24 The Trustees Of Columbia University In The City Of New York Expression of enzymatically active reverse transcriptase
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5017492A (en) 1986-02-27 1991-05-21 Life Technologies, Inc. Reverse transcriptase and method for its production
WO1992006200A1 (fr) 1990-09-28 1992-04-16 F. Hoffmann-La-Roche Ag Mutations d'adn-polymerases thermostables en 5' a 3' exonuclease
US5244797A (en) 1988-01-13 1993-09-14 Life Technologies, Inc. Cloned genes encoding reverse transcriptase lacking RNase H activity
US5374553A (en) 1986-08-22 1994-12-20 Hoffmann-La Roche Inc. DNA encoding a thermostable nucleic acid polymerase enzyme from thermotoga maritima
US5455170A (en) 1986-08-22 1995-10-03 Hoffmann-La Roche Inc. Mutated thermostable nucleic acid polymerase enzyme from Thermus species Z05
US5466591A (en) 1986-08-22 1995-11-14 Hoffmann-La Roche Inc. 5' to 3' exonuclease mutations of thermostable DNA polymerases
WO1996010640A1 (fr) 1994-09-30 1996-04-11 Life Technologies, Inc. Adn-polymerases clone a partir de thermotoga neapolitana et leurs mutants
US5538848A (en) 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
WO1998047912A1 (fr) 1997-04-22 1998-10-29 Life Technologies, Inc. Procedes de production de transcriptases inverse aslv constituees de sous-unites multiples
WO1999021881A1 (fr) 1997-10-27 1999-05-06 Boston Probes, Inc. Procedes, trousses et compositions relatifs a des balises lineaires
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
US6200757B1 (en) 1999-01-19 2001-03-13 Dade Behring Inc. Method for controlling the extension of an oligonucleotide
US6210882B1 (en) 1998-01-29 2001-04-03 Mayo Foundation For Medical Education And Reseach Rapid thermocycling for sample analysis
US6303305B1 (en) 1999-03-30 2001-10-16 Roche Diagnostics, Gmbh Method for quantification of an analyte
US6355421B1 (en) 1997-10-27 2002-03-12 Boston Probes, Inc. Methods, kits and compositions pertaining to PNA molecular beacons
US6383752B1 (en) 1999-03-31 2002-05-07 Hybridon, Inc. Pseudo-cyclic oligonucleobases
US6403341B1 (en) 2001-08-02 2002-06-11 Wayne M. Barnes Magnesium precipitate hot start method for PCR
US6548250B1 (en) 1999-10-29 2003-04-15 Stratagene Methods for detection of a target nucleic acid sequence
US6590091B2 (en) 1994-12-27 2003-07-08 Naxcor Nucleic acid sequence detection employing amplification probes
US6589743B2 (en) 2000-10-11 2003-07-08 Stratagene Methods for detection of a target nucleic acid using a probe comprising secondary structure
US6589250B2 (en) 2001-11-20 2003-07-08 Stephen A. Schendel Maxillary distraction device
US6593091B2 (en) 2001-09-24 2003-07-15 Beckman Coulter, Inc. Oligonucleotide probes for detecting nucleic acids through changes in flourescence resonance energy transfer
US6596490B2 (en) 2000-07-14 2003-07-22 Applied Gene Technologies, Inc. Nucleic acid hairpin probes and uses thereof
US6727356B1 (en) 1999-12-08 2004-04-27 Epoch Pharmaceuticals, Inc. Fluorescent quenching detection reagents and methods
US20040096819A1 (en) 2000-05-01 2004-05-20 Cepheid Method for quantitative analysis of a nucleic acid amplification reaction
US7056716B2 (en) 2000-03-15 2006-06-06 Invitrogen Corporation High fidelity reverse transcriptases and uses thereof
US7078208B2 (en) 2000-05-26 2006-07-18 Invitrogen Corporation Thermostable reverse transcriptases and uses thereof
US7228237B2 (en) 2002-02-07 2007-06-05 Applera Corporation Automatic threshold setting and baseline determination for real-time PCR
WO2008077017A2 (fr) * 2006-12-19 2008-06-26 Sigma-Aldrich Co. Compositions stabilisées d'adn polymérase thermostable et détergent anionique ou zwittérionique
US20090197254A1 (en) 2007-12-14 2009-08-06 Ming-Chou Lee Variant scorpion primers for nucleic acid amplification and detection
US20090269766A1 (en) 2008-03-19 2009-10-29 Dieter Heindl Nucleic acid amplification in the presence of modified randomers
US8689807B2 (en) 2012-03-06 2014-04-08 Svenson Co., Ltd. Wig clip
US8689818B2 (en) 2008-06-25 2014-04-08 Masco Corporation Of Indiana Widespread faucet
US9005895B2 (en) * 2010-06-21 2015-04-14 Life Technologies Corporation Compositions, methods and kits for nucleic acid synthesis and amplification
WO2017177025A1 (fr) * 2016-04-06 2017-10-12 Life Technologies Corporation Compositions, procédés, et kits pour la synthèse et la détection d'acides nucléiques
EP3257952B1 (fr) * 2012-09-11 2020-02-12 Life Technologies Corporation Amplification d'acides nucléiques
CN116004776A (zh) * 2022-08-30 2023-04-25 上海伯杰医疗科技股份有限公司 一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法

Patent Citations (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4943531A (en) 1985-05-06 1990-07-24 The Trustees Of Columbia University In The City Of New York Expression of enzymatically active reverse transcriptase
US5017492A (en) 1986-02-27 1991-05-21 Life Technologies, Inc. Reverse transcriptase and method for its production
US5455170A (en) 1986-08-22 1995-10-03 Hoffmann-La Roche Inc. Mutated thermostable nucleic acid polymerase enzyme from Thermus species Z05
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4889818A (en) 1986-08-22 1989-12-26 Cetus Corporation Purified thermostable enzyme
US5466591A (en) 1986-08-22 1995-11-14 Hoffmann-La Roche Inc. 5' to 3' exonuclease mutations of thermostable DNA polymerases
US5374553A (en) 1986-08-22 1994-12-20 Hoffmann-La Roche Inc. DNA encoding a thermostable nucleic acid polymerase enzyme from thermotoga maritima
US5244797B1 (en) 1988-01-13 1998-08-25 Life Technologies Inc Cloned genes encoding reverse transcriptase lacking rnase h activity
US5244797A (en) 1988-01-13 1993-09-14 Life Technologies, Inc. Cloned genes encoding reverse transcriptase lacking RNase H activity
US5668005A (en) 1988-01-13 1997-09-16 Life Technologies, Inc. Cloned genes encoding reverse transcriptase lacking RNASE H activity
WO1992006200A1 (fr) 1990-09-28 1992-04-16 F. Hoffmann-La-Roche Ag Mutations d'adn-polymerases thermostables en 5' a 3' exonuclease
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US6103476A (en) 1993-11-12 2000-08-15 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled, dual conformation oligonucleotide probes, assays and kits
WO1996010640A1 (fr) 1994-09-30 1996-04-11 Life Technologies, Inc. Adn-polymerases clone a partir de thermotoga neapolitana et leurs mutants
US5912155A (en) 1994-09-30 1999-06-15 Life Technologies, Inc. Cloned DNA polymerases from Thermotoga neapolitana
US5538848A (en) 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
US6590091B2 (en) 1994-12-27 2003-07-08 Naxcor Nucleic acid sequence detection employing amplification probes
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
WO1998047912A1 (fr) 1997-04-22 1998-10-29 Life Technologies, Inc. Procedes de production de transcriptases inverse aslv constituees de sous-unites multiples
WO1999021881A1 (fr) 1997-10-27 1999-05-06 Boston Probes, Inc. Procedes, trousses et compositions relatifs a des balises lineaires
US6355421B1 (en) 1997-10-27 2002-03-12 Boston Probes, Inc. Methods, kits and compositions pertaining to PNA molecular beacons
US6485901B1 (en) 1997-10-27 2002-11-26 Boston Probes, Inc. Methods, kits and compositions pertaining to linear beacons
US6210882B1 (en) 1998-01-29 2001-04-03 Mayo Foundation For Medical Education And Reseach Rapid thermocycling for sample analysis
US6200757B1 (en) 1999-01-19 2001-03-13 Dade Behring Inc. Method for controlling the extension of an oligonucleotide
US6303305B1 (en) 1999-03-30 2001-10-16 Roche Diagnostics, Gmbh Method for quantification of an analyte
US6503720B2 (en) 1999-03-30 2003-01-07 Roche Diagnostics Gmbh Method for quantification of an analyte
US6383752B1 (en) 1999-03-31 2002-05-07 Hybridon, Inc. Pseudo-cyclic oligonucleobases
US6548250B1 (en) 1999-10-29 2003-04-15 Stratagene Methods for detection of a target nucleic acid sequence
US6727356B1 (en) 1999-12-08 2004-04-27 Epoch Pharmaceuticals, Inc. Fluorescent quenching detection reagents and methods
US7056716B2 (en) 2000-03-15 2006-06-06 Invitrogen Corporation High fidelity reverse transcriptases and uses thereof
US20040096819A1 (en) 2000-05-01 2004-05-20 Cepheid Method for quantitative analysis of a nucleic acid amplification reaction
US6783934B1 (en) 2000-05-01 2004-08-31 Cepheid, Inc. Methods for quantitative analysis of nucleic acid amplification reaction
US7078208B2 (en) 2000-05-26 2006-07-18 Invitrogen Corporation Thermostable reverse transcriptases and uses thereof
US6596490B2 (en) 2000-07-14 2003-07-22 Applied Gene Technologies, Inc. Nucleic acid hairpin probes and uses thereof
US6589743B2 (en) 2000-10-11 2003-07-08 Stratagene Methods for detection of a target nucleic acid using a probe comprising secondary structure
US6403341B1 (en) 2001-08-02 2002-06-11 Wayne M. Barnes Magnesium precipitate hot start method for PCR
US6593091B2 (en) 2001-09-24 2003-07-15 Beckman Coulter, Inc. Oligonucleotide probes for detecting nucleic acids through changes in flourescence resonance energy transfer
US6589250B2 (en) 2001-11-20 2003-07-08 Stephen A. Schendel Maxillary distraction device
US7228237B2 (en) 2002-02-07 2007-06-05 Applera Corporation Automatic threshold setting and baseline determination for real-time PCR
WO2008077017A2 (fr) * 2006-12-19 2008-06-26 Sigma-Aldrich Co. Compositions stabilisées d'adn polymérase thermostable et détergent anionique ou zwittérionique
US20090197254A1 (en) 2007-12-14 2009-08-06 Ming-Chou Lee Variant scorpion primers for nucleic acid amplification and detection
US20090269766A1 (en) 2008-03-19 2009-10-29 Dieter Heindl Nucleic acid amplification in the presence of modified randomers
US8689818B2 (en) 2008-06-25 2014-04-08 Masco Corporation Of Indiana Widespread faucet
US9005895B2 (en) * 2010-06-21 2015-04-14 Life Technologies Corporation Compositions, methods and kits for nucleic acid synthesis and amplification
US8689807B2 (en) 2012-03-06 2014-04-08 Svenson Co., Ltd. Wig clip
EP3257952B1 (fr) * 2012-09-11 2020-02-12 Life Technologies Corporation Amplification d'acides nucléiques
WO2017177025A1 (fr) * 2016-04-06 2017-10-12 Life Technologies Corporation Compositions, procédés, et kits pour la synthèse et la détection d'acides nucléiques
CN116004776A (zh) * 2022-08-30 2023-04-25 上海伯杰医疗科技股份有限公司 一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
BROUDE ET AL., TRENDS BIOTECHNOL, vol. 20, 2002, pages 249 - 56
CHIOU ET AL., ANAL. CHEM., vol. 73, pages 2018 - 2021
FLAMAN ET AL., NUCL. ACIDS RES, vol. 219, 1994, pages 3259 - 3260
GERARD ET AL., FOCUS, vol. 14, 1992, pages 91 - 93
GIORDANO ET AL., ANAL. BIOCHEM, vol. 291, 2001, pages 124 - 132
HUANG ET AL., CHEM RES. TOXICOL., vol. 15, 2002, pages 118 - 126
ISACSSON ET AL., MOLECULAR CELL PROBES, vol. 14, 2000, pages 321 - 328
KALININA ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 1999 - 2004
KOPP ET AL., SCIENCE, vol. 280, 1998, pages 1046 - 1048
KOTEWICZ ET AL., NUCL. ACIDS RES, vol. 16, 1988, pages 265 - 277
KUBISTA ET AL., PROCEEDINGS IN SPIE, vol. 4264, 2001, pages 53 - 58
MAXWELL ET AL., J. AM. CHEM. SOC., vol. 124, 2002, pages 9606 - 9612
MHLANGA ET AL., METHODS, vol. 25, 2001, pages 463 - 471
MYERSGELFAND, BIOCHEMISTRY, vol. 30, 1991, pages 7661 - 7666
PERBAL: "A Practical Guide to Molecular Cloning", 1984, WILEY & SONS, pages: 23 - 24
RICCELLI ET AL., NUCLEIC ACIDS RESEARCH, vol. 30, 2002, pages 4088 - 4093
SELLNERTURBETT, BIOTECHNIQUES, vol. 25, 1998, pages 234 - 238
SOLINAS ET AL., NUCLEIC ACIDS RESEARCH, vol. 29, 2001, pages E96
SVANVIK ET AL., ANAL. BIOCHEM, vol. 281, 2000, pages 26 - 25
TYAGIKRAMER, NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 303 - 308
WHITCOMBE ET AL., NATURE BIOTECHNOLOGY, vol. 17, 1999, pages 804 - 807
WOLFFS ET AL., BIOTECHNIQUES, vol. 766, 2001, pages 769 - 771
YU ET AL., J. AM. CHEM. SOC., vol. 14, 2001, pages 11155 - 11161
ZHANG ET AL., SHANGHAI, vol. 34, 2002, pages 329 - 332

Similar Documents

Publication Publication Date Title
US20210292812A1 (en) Compositions, methods and kits for nucleic acid synthesis and amplification
US12054790B2 (en) Compositions, methods and kits for microorganism detection
RU2757416C2 (ru) Композиции, способы и наборы для синтеза и обнаружения нуклеиновых кислот
JP7490022B2 (ja) ポリメラーゼ連鎖反応（ｐｃｒ）のための新規の組成物、方法、およびキット
US20230313286A1 (en) Nucleic acid extraction and amplification controls and methods of use thereof
WO2025007089A1 (fr) Compositions, méthodes et kits de synthèse et d'amplification d'acides nucléiques
WO2025175212A1 (fr) Procédés de génotypage sans extraction rapide
WO2025175243A1 (fr) Protocole d'amplification et de collecte de données pour génotypage rapide

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24748728

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE