WO2008157689A9 - Procédés d'analyse d'amplification d'acides nucléiques - Google Patents

Procédés d'analyse d'amplification d'acides nucléiques Download PDF

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WO2008157689A9
WO2008157689A9 PCT/US2008/067496 US2008067496W WO2008157689A9 WO 2008157689 A9 WO2008157689 A9 WO 2008157689A9 US 2008067496 W US2008067496 W US 2008067496W WO 2008157689 A9 WO2008157689 A9 WO 2008157689A9
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amplification
nucleic acid
data
target nucleic
target
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PCT/US2008/067496
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WO2008157689A2 (fr
WO2008157689A3 (fr
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Alexander M Chagovetz
James P Keener
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Univ Utah Res Found
Alexander M Chagovetz
James P Keener
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Publication of WO2008157689A3 publication Critical patent/WO2008157689A3/fr

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    • 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

  • the disclosed invention is generally in the field of biotechnology, more specifically to the field of nucleic acid amplification analysis.
  • PCR polymerase chain reaction
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • RTD-QPCR Real-time detection quantitative PCR
  • non-specific systems are fluorophores that can bind to double-stranded sequences of nucleic acids based on common structural features of all nucleic acids.
  • the dye is an intercalating fluorescent dye, or a minor groove binding dye, which exhibits fluorescence upon binding to a double-stranded amplification product on direct excitation with light.
  • the dye has different fluorescence efficiency (quantum yields of fluorescence) depending on the bound or unbound (free) state of a fluorophore.
  • a dye is introduced into the PCR reaction volume, with the primers, and other reaction components, prior to reaction. The dye then binds to the double stranded amplification products as they are formed in solution and the characteristic signal of the dye is measured as the reaction progresses. Examples of dyes used for this purpose include SYBR GREEN I ® (Molecular Probes, Oregon) and ethidium bromide.
  • FRET fluorescence resonance energy transfer
  • a common approach is to employ FRET techniques with probe technology to detect and monitor DNA amplification.
  • Fluorescent dye labels typically are small organic dye molecules, such as fluorescein, Texas red, or rhodamine, which can be readily conjugated to probe-type molecules.
  • the fluorescent molecules can be detected by illumination with light of an appropriate frequency. Light excites the fluorophores and produces a resultant emission spectrum that can be detected by electro-optical sensors or light microscopy.
  • FRET occurs between a donor fluorophore and an acceptor dye, which may be a fluorophore, when the donor fluorophore has an emission spectrum that overlaps the absorption spectrum of the acceptor dye, and the donor fluorophore and acceptor dye are in sufficiently close physical proximity.
  • an acceptor dye which may be a fluorophore
  • the donor fluorophore has an emission spectrum that overlaps the absorption spectrum of the acceptor dye, and the donor fluorophore and acceptor dye are in sufficiently close physical proximity.
  • an acceptorophore When light excites the donor fluorophore, there is then produced an emission of light that may be absorbed and quenched by the acceptor molecule.
  • quenching occurs, the intensity of the donor fluorophore's emission appears to be lessened.
  • the acceptor is also a fluorophore, the intensity of its fluorescence may be enhanced.
  • the efficiency of energy transfer is highly dependent on the distance between the donor and acceptor, and equations
  • FRET is a function of the distance between the donor and acceptor molecules.
  • a discussion of these relationships and Forster - type equations is found Parkhurst and Parkhurst, Donor-acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes, Biochemistry, 34: 293-300 (1995), the contents of the entirety of which are incorporated by this reference.
  • TAQMAN ® assay uses a hydrolysis probe labeled with donor fluorophore and acceptor dye, which is then cleaved by the 5' to 3' exonuclease activity of the enzyme Taq polymerase to cause an increase in the intensity of the donor fluorophore.
  • the probe is labeled with both donor and acceptor, and (prior to the attachment of the probe to a DNA strand) the fluorescence of the donor is quenched by the acceptor.
  • the probe is hybridized to the DNA strand to be amplified.
  • the 5' to 3' exonuclease activity of the polymerase causes cleavage of the probe, separating the donor and acceptor and resulting in an increase in intensity of the fluorescence of the donor fluorophore.
  • Woo et ai Identification of pathogenic Leptospira by TaqMan probe in a LightCycler, Anal. Biochem., 256: 132-134 (1998), the contents of the entirety of which are incorporated by this reference.
  • Another method for detecting amplification products is the "molecular beacon probe.”
  • This method uses oligonucleotide hybridization probes that form hairpin structures, with the donor fluorophore on the 5' end, and the acceptor molecule on the 3 * end of the hybridization probe. When the probe is in a hairpin conformation, the donor and acceptor are in close proximity, and the fluorescence of the donor fluorophore is quenched.
  • the molecular beacon probe hybridizes to one of the strands of the PCR product, and is in "open conformation" such that the donor fluorophore and acceptor dye are separated and the fluorescence intensity of the donor increases to a level that can be detected. See Tyagi and Kramer, Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol, 14: 303-308 (1996), the contents of the entirety of which are incorporated by this reference.
  • PCR may be carried out with a primer labeled with the fluorophore CY5 in the presence of a fluorescein-labeled probe.
  • the CY5 primer is attached to the target sequence and is used to form an extension product.
  • the fluorescein-labeled probe then hybridizes to the extension product, the CY5 strand.
  • the fluorophores of the primer and probe are in close proximity, and resonance energy transfer occurs between the fluorophores, increasing the fluorescence of the CY5.
  • observation of fluorescence and, in particular, the FRET signal allows monitoring of the hybridization of probe to target, and melting of the probe away from the target, during melting curve analysis of the probe away from the target.
  • the methods described above rely upon the efficiency of the hybridization of the probe to the target. If the probe does not efficiently hybridize to the target sequence, the intensity of the generated signal, used to measure the quantity of the amplification product, is affected. Additionally, the probes may interfere with the nucleic acid amplification process. When a probe binds to the template strand of nucleic acid, it converts a piece of single-stranded template into double-stranded helix. The probe blocks the nucleic acid polymerase from completing translocation along the nucleic acid strand halting replication until the probe is removed, either by melting or enzyme action.
  • a method that relies not upon probes, but only upon applying fluorescent dye labels to a single oligonucleotide primer molecule in a hairpin structure has been described as an approach for detecting the presence of a target nucleic acid sequence and the quantity of such nucleic acid sequence in a sample.
  • the primer is designed to have two dye labels on the stem of its hairpin structure.
  • One label is a fluorophore donor, and the other is a quencher that absorbs energy emitted by the donor.
  • the fluorophore donor and acceptor are in close proximity, so that the fluorescence of the donor is substantially quenched by the acceptor.
  • the hairpin structure is linearized, a complementary strand is synthesized, and the primer with its fluorescent labels is incorporated into the amplification product.
  • the primer is opened and incorporated into the product, the fluorophore donor and acceptor become widely separated, reducing the quenching effect.
  • An advantage of this method is that the fluorescent signal is generated by the product itself, and not through the use of a probe.
  • the use of a hairpin primer typically involves several difficulties, including design of an awkward, long primer having a hairpin configuration, which may not be easily “read” by the polymerase, and the need to place two labels, donor and acceptor, on the hairpin primer in specific locations.
  • the hairpin primer may present other disadvantages, including competition of formation of the hairpin with formation of double-stranded DNA (resulting in lower sensitivity in detection of the FRET signal) and potential formation of primer-dimers, which may interfere with detection of the product signal. Also, the stability of the hairpin has to be low enough to allow enzymatic read through, but high enough to reduce background fluorescence, which creates an inherent contradiction that may reflect on sensitivity of the assay. In a further development, the hairpin primers may be designed to have only one fluorescent tag in the proximity of 3 'end using the quenching effect of adjacent G moieties.
  • RTD-PCR real-time detection PCR
  • threshold cycle is defined as a time point corresponding to the maximal value of the second derivative, i.e., at the point where the fluorescent signal starts growing from the baseline fluorescence.
  • numerical derivatization is substituted by derivatization of the analytical fit (spline function) to experimental data (LCDA 3.5, Roche Diagnostics), which results in improved accuracy of quantitative results.
  • Embodiments of the invention include an apparatus for quantitatively determining an amount of nucleic acid in a sample.
  • the apparatus includes a means for describing amplification of nucleic acids, as an iterative process, from one cycle to a next cycle in a nucleic acid amplification method.
  • x n is a relative quantity of nucleic acid present at a given point of a first nucleic acid amplification cycle
  • x n +i is a relative quantity of nucleic acid present at the same given point of a second said nucleic acid amplification cycle
  • f(x n ) is a growth function based upon a kinetic model of a chosen method of amplification and a chosen detection chemistry.
  • the computer-readable medium may be further programmed to receive data generated by the chosen detection chemistry and to iteratively fit the equation to the data.
  • Embodiments of the invention include a method for quantifying a target nucleic acid in a sample.
  • the method may include acquiring amplification data from real-time detection ("RTD") of amplification of the target nucleic acid.
  • RTD real-time detection
  • a first iterative growth function may be fit to the acquired amplification data.
  • the fitted first iterative growth function may be reconstructed to the beginning of the amplification reaction to calculate a relative initial target quantity in the sample.
  • FIG. 1 illustrates raw fluorescence data collected during real-time detection polymerase chain reaction (“RTD-PCR") of thirteen different samples.
  • RTD-PCR real-time detection polymerase chain reaction
  • FIG. 2A illustrates raw primer-dimer data from two samples.
  • FIG. 2B illustrates the primer-dimer data of FIG. 2 A after the data has been cut and scaled.
  • FIG. 3 A in the upper illustrates the raw data of FIG. 1 prior to being cut and scaled.
  • FIG. 3 A in the lower panel illustrates the raw data of FIG. 1 after one embodiment of cutting and scaling the raw data.
  • FIG. 3B illustrates one embodiment of functions fit to the cut and scaled data of the lower panel of FIG. 3 A.
  • FIGS. 3 C and 3D illustrate the first and second derivative, respectively, of the functions illustrated in FIG. 3B.
  • FlG. 4A illustrates the functions which serves as the polynomial basis for the spline functions of FIG. 3B.
  • FIGS. 4B and 4C illustrate the first and second derivative, respectively, of the functions illustrated in FIG. 4A.
  • FIGS. 4D and 4E illustrate embodiments of filtering processes used to identify data that did not show significant growth.
  • FIG. 4F illustrates the fit target data of FIG. 4E after one embodiment of scaling the filtered target data.
  • FIG. 5 illustrates one embodiment of system specific scaling for the SYBR GREEN I ® detection system.
  • FIG. 6A illustrates raw data collected using a hybridization probe detection system.
  • FIG. 6B illustrates the hybridization probe raw data after one embodiment of cutting and scaling the data.
  • FIG. 6C illustrates one embodiment of determining relative quantitative amounts of target DNA ("x n ”) based on the cut and scaled data of FIG. 6B.
  • FIGS. 6D and 6E illustrate the derivatives of the data of FIG. 6C with respect to the fluorescence signal ("d n ”) and with respect to the scaling factor ("s"), respectively, after a single iteration.
  • FIG. 6F illustrates one embodiment of a calculated relationship between d n and x n .
  • FIG. 7 illustrates one embodiment of estimated growth functions based on calculated relative initial quantities of target DNA.
  • FIG. 8 A illustrates one embodiment of estimated growth curves for the primer- dimer data of FIG. 2 A.
  • FIG. 8B illustrates one embodiment of estimated growth curves for target DNA data of FIG. 1.
  • FIG. 8C illustrates an estimated target DNA ("template”) growth, estimated primer-dimer growth, estimated target DNA efficiency, and estimated primer-dimer efficiency.
  • FIG. 9A illustrates a calibration curve generated using the SYBR GREEN I ® detection system and iterative growth functions.
  • FIG. 9B illustrates estimated target DNA growth and efficiency using the SYBR GREEN I ® data of FIG. 9A.
  • FIG. 9C illustrates the relative initial quantities used to generate the calibration curve ofFIG. 9A.
  • FIG. 1 OA illustrates a calibration curve generated using a prior art second derivative maximum method and the same SYBR GREEN I ® data used to generate the calibration curve of FlG. 9A.
  • FIG. 1 OB illustrates quantification verification of the second derivative maximum method data.
  • the upper left panel of FIG. 1 IA illustrates the raw data illustrated in FIG. 6 A.
  • the upper right panel illustrates the scaled data illustrated in FIG. 6B.
  • the bottom panel illustrates raw SYBR GREEN I ® negative data.
  • the upper left panel of FIG. 1 IB illustrates estimated target DNA growth based on the data of FIG. 1 IA.
  • the upper right panel illustrates estimated primer-dimer growth based upon the data of FIG. 1 IA.
  • the bottom left panel illustrates estimated target DNA efficiency over the course of amplification.
  • the bottom right panel illustrates estimated primer-dimer efficiency over the course of amplification.
  • the upper left panel of FIG. 11C illustrates the calculated relative initial quantities that were used with known initial quantities to generate the calibration curve illustrated in the upper right panel.
  • the bottom left and right panels illustrate respectively quantification verification and results using iteratively fit growth functions.
  • FIG. 12 illustrates in the upper left panel the same raw data illustrated in FIG. 1 IA.
  • FIG. 12 illustrates in the upper right panel a calibration curve generated using a prior art second derivative maximum method.
  • the bottom left and right panels illustrate respectively quantification verification and results using the second derivative maximum method data.
  • FIG. 13 in the upper left panel, illustrates raw fluorescence data from RTD-PCR using TAQMAN ® probes and lambda DNA with an internal control.
  • the upper right panel illustrates just the fluorescence data from the lambda DNA growth.
  • the bottom left and right panels illustrate respectively calculated target DNA efficiency and internal control efficiency.
  • FIG. 14 illustrates a calibration curve generated using the data illustrated in the upper left panel of FIG. 13 and the direct method.
  • FIG. 14 also illustrates the data used to generate the calibration curve and quantification verification and results based on that data.
  • FIG. 15 illustrates a comparison between one embodiment of a direct method and one embodiment of an indirect method using the data illustrated in the upper left panel of FIG. 13.
  • FIG. 16 illustrates a calibration curve generated using the indirect method.
  • FIG. 16 also illustrates the data used to generate the calibration curve and quantification verification and results based on that data.
  • Embodiments of the invention may apply to quantitative analysis of target nucleic acid amplification.
  • each amplification reaction is presumed to be a multiplex reaction. Even with a single pair of primers there are at least two concurrent reactions: specific reaction (e.g., target nucleic acid amplification) and non-specific reaction (e.g., primer-dimer formation). Additionally, there may be a competitive nucleic acid (e.g., an internal control) competing with the target nucleic acid for primers. Every reaction in each amplification cycle, at least partially, competes with all other reactions (e.g., competition for primers, nucleotides, enzymes, Mg, hydration, hybridization, etc.).
  • Competition is manifested in the amplification efficiency, even if one or more reactions are "detection silent" (i.e., increased competition reduces target nucleic acid amplification and thereby reduces the amplification efficiency). This, in general, results in varying amplification efficiency for each reaction in each amplification cycle.
  • Embodiments of the invention include acquiring amplification data from real-time detection (“RTD”) of the amplification method.
  • RTD real-time detection
  • PCR Polymerase Chain Reaction
  • SDA strand displacement amplification
  • SPA single primer amplification
  • HAD helicase-dependent amplification
  • Amplification data may include primary fluorescence data.
  • amplification data may be generated with intercalating dyes, with labeled primers, labeled primers and probes, hybridization probes, or hydrolysis probes.
  • Embodiments of the invention are not limited by the type of nucleic acid amplified.
  • deoxyribonucleic acids (“DNA”) are primarily referred to as the target nucleic acids; however, any nucleic acid that may be detectably amplified may be analyzed with embodiments of the invention.
  • RNA may be reversibly transcribed to form complementary DNA (“cDNA”) and then the cDNA amplified.
  • Embodiments of the invention may include a means for describing amplification of nucleic acids, as an iterative process, from one cycle to a next cycle in a nucleic acid amplification method.
  • the means for describing amplification of nucleic acids may take into consideration both target nucleic acid amplification and specific or non-specific amplifications that compete with target nucleic acid amplification.
  • the means for describing amplification of nucleic acids may include the equation:
  • X n is a relative quantity of nucleic acid present at a given point of a first nucleic acid amplification cycle
  • x n +i is a relative quantity of nucleic acid present at the same given point of a second said nucleic acid amplification cycle
  • f(x n ) is a growth function based upon a kinetic model of a chosen method of amplification and a chosen detection chemistry.
  • the growth functions may be based upon a kinetic model for the particular amplification method used and the particular detection chemistry used.
  • the growth functions may be iteratively fit to the amplification data. Relative initial target nucleic acid amounts may then be calculated using the fitted growth functions.
  • the efficiency of target nucleic acid amplification and competing amplification may also be determined from the fitted growth functions.
  • the iterative growth functions may also be used to generate calibration curves.
  • Known initial quantities of target nucleic acids may be amplified using RTD-PCR. Iterative growth functions may be fit to the RTD-PCR data and then relative initial quantities of the target DNA calculated.
  • Calibration curves may be generated that are specific to a particular type of PCR apparatus and even specific to individual units. Additionally, calibration curves may be generated that are specific to particular primers and target DNA.
  • Fluorescence intensity may be correlated with the concentration of the target nucleic acids, and within a broad range of concentrations (early-to-mid cycles of PCR) this correlation is monotonic.
  • the scaling may result in a transformation of the detected fluorescence intensity into relative concentrations of amplified target nucleic acid if the maximum fluorescence intensity corresponds to the maximum target nucleic acid concentration.
  • the growth functions may then be iteratively fit to the scaled data.
  • the means for describing amplification of target nucleic acids may account for the effects of competition for target nucleic acid amplification with the amplification of non-specific products, such as primer-dimers. Accounting for the effects of competition may involve amplifying a non-specific material in the absence of target nucleic acids, wherein the non-specific material is ordinarily present when the target nucleic acid is present in a sample.
  • the primers to be used with the target nucleic acid may be run through RTD-PCR without any target nucleic acid present. Therefore, primer-dimers would be amplified.
  • non-specific amplification data from real-time detection ("RTD") of amplification of the non-specific material may be acquired (e.g., fluorescence data collected). Growth functions may then be iteratively fit to the acquired non-specific amplification data. The fitted growth functions may be reconstructed to calculate a relative initial quantity of non-specific material.
  • the fitted non-specific growth functions may be used with fitted target nucleic acid growth functions (i.e., growth functions fit to target nucleic acid amplification data) to construct new target nucleic acid growth functions and new non-specific material growth functions.
  • the new target nucleic acid growth functions may be included in the means for describing amplification of the target nucleic acids.
  • Embodiments of the invention may include amplifying a specific competing nucleic acid of known initial quantity while amplifying the target nucleic acid.
  • the means for describing amplification of target nucleic acids may account for the effects of competition for target nucleic acid amplification with the amplification of competing nucleic acids, such as internal controls. Accounting for the effects of competition may involve amplifying a competing nucleic acid in the absence of target nucleic acids. For example, an internal control to be used with the target nucleic acid, may be run through RTD-PCR without any target nucleic acid present. Therefore, the internal control would be amplified.
  • amplification data from real-time detection RTD of amplification of the competing nucleic acid may be acquired (e.g., fluorescence data collected). Growth functions may then be iteratively fit to the acquired competing nucleic acid amplification data. The fitted growth functions may be reconstructed to calculate a relative initial quantity of the competing nucleic acid.
  • the fitted competing nucleic acid functions may be used with fitted target nucleic acid growth functions (i.e., growth functions fit to target nucleic acid amplification data) to construct new target nucleic acid growth functions and competing nucleic acid growth functions.
  • the new target nucleic acid growth functions may be included in the means for describing amplification of the target nucleic acids.
  • Internal controls may be useful in medical diagnostic applications where it may be undesirable to use external calibration.
  • the use of internal controls may increase the accuracy of calibration curves.
  • Internal controls may be used in non-medical diagnsostic applications as well.
  • the internal control may be a known amount of a nucleic acid that will compete with the target nucleic acid for amplification.
  • Internal control data may be used in a "direct” method or an “indirect” method.
  • the direct method utilizes signals from both target DNA and internal control during target DNA amplification.
  • the indirect method only uses signals from the internal control during target DNA amplification.
  • the direct and indirect method may also be used with primer-dimer growth data or any other competing growth data.
  • the means for describing amplification of target nucleic acids may be used to analyze fluorescence intensity data from RTD-PCR.
  • RTD-PCR may use fluorescent detection to identify when amplification has occurred. As the amount of target nucleic acid is increased by amplification, the intensity of fluorescence increases. The fluorescence is tracked cycle by cycle. Generally, RTD-PCR proceeds for about 40 cycles. After about 40 cycles the amount of DNA may have stopped amplifying, so that more cycles do not result in substantially more DNA. The fluorescence per cycle (hereinafter the "raw amplification data”) may be analyzed with embodiments of the invention.
  • the raw amplification data output by the RTD-PCR system may be in terms of fluorescent intensity per cycle. Numerous samples of a given target nucleic acid may be amplified. Each sample may be individually amplified in a separate RTD-PCR tube.
  • FIG. 1 illustrates the raw fluorescence data collected during RTD-PCR of thirteen different samples.
  • FIG. 1 represents the raw amplification data of each sample as a curved line for convenience. However, it should be understood that each sample may be represented as a collection of points, without interconnecting lines between the points. It should also be understood that different detection chemistries generate amplification data at different points during each cycle. For example, when an intercalating dye is used, then fluorescence intensity will be greatest when target nucleic acids are hybridized as double-stranded DNA. Thus, fluorescence intensity will be greatest at the end of each cycle (if the cycle starts with anneal step this is not true).
  • FIG. 1 represents the peak fluorescence per cycle for each of the samples.
  • the increase in fluorescence per cycle is proportional to the increase in the amount of target nucleic acids in each of the samples.
  • the principal difference between TAQMAN ® and the rest of the chemistries is that in the former case the signal is accumulated from cycle to cycle (i.e., the signal is "indestructible” because hydrolysis of the probes is irreversible), while for the latter cases the signal is generated in each cycle de novo (i.e., starting with zero at the melt point).
  • Embodiments of the invention may be incorporated in an apparatus for either conducting RTD-PCR or for analyzing data from RTD-PCR. Embodiments of the invention may be included in a software program for analyzing RTD-PCR data. Embodiments of the invention may also be used as an improvement or upgrade to existing RTD-PCR analysis software. Embodiments of the invention include a method of doing business where RTD-PCR data analysis is performed as a service.
  • the growth functions are second degree polynomials.
  • the second degree polynomials may then be fit to the amplification data.
  • Examples 1 and 2 provide a theoretical basis for fitting second degree polynomials as the growth functions.
  • ss target DNA single-stranded target DNA
  • ds target DNA double-strand target DNA
  • T is ss target DNA
  • P primer
  • Il product (ds target DNA).
  • Equation (4) is a linear differential equation, and hence solvable with an integrating factor, yielding:
  • TQ and PQ are the initial amounts of ss target DNA and primer at lhe beginning of a given cycle, respectively. Assuming the reaction runs to completion for that cycle, K may be solved from equation (5) yielding:
  • Equation (9) may also be written as:
  • ⁇ 7 -k ⁇ PT- k ⁇ - k ⁇ RT, (12) dt dP_ Ic 1 PT, (13) dt dR_ ⁇ k,RT, (14) dt M -k x PT+ k 1 T 1 . (15) dt
  • Equations (12) - (16) may be viewed as functions of primers:
  • the constants K ⁇ and .K 2 may be determined from initial data according to:
  • the signal is an indicator of ss target DNA at the beginning of a given cycle.
  • Equation (33) may be iteratively fit to a target DNA growth function to solve for X. For example only, if after fitting equation (33) to the target DNA growth function the following function results,
  • Equations (10) and (26) are only examples of kinetic models including growth functions. Examples 1 and 2 include the assumptions discussed above. Variations in assumptions will result in different growth functions. Additionally, variations in detection chemistry may result in different growth functions. Different detection systems generate different functional dependences of fluorescence intensity versus target concentrations. These functions are combinations of amplification efficiencies and detection efficiencies (hence they are detection system specific). Data generation and processing with internal controls is similar to when primer- dimer competition is being analyzed. Kinetic models and growth functions may be generated, similar to that in Examples 1 and 2, which account for competition between the internal control and the target DNA. The primer-dimer kinetic equations in Examples 1 and 2 may be replaced with internal control equations.
  • primer-dimers may still form during target DNA and/or internal control amplification.
  • the competition from primer-dimer formation may be lumped in with the competition from the internal control.
  • the data collected from RTD-PCR with only the internal control may be considered to show both internal control DNA amplification as well as primer-dimer amplification.
  • the growth curves iteratively fit to the internal control data may also be used to take into account the effects of primer-dimer amplification on target DNA amplification.
  • any other competing factor during target DNA amplification may be lumped into the competing effects of either internal control amplification or primer-dimer amplification.
  • the above models may form the basis for quantification analysis. Additionally, other competing reactions may also be added to or substituted into the model in a similar manner.
  • Lambda DNA approximately 150 base pairs in length, was amplified using a RTD-PCR Roche Applied Science Lightcycler ® 2.0. Amplifications were conducted using SYBR GREEN I ® , hybridization probes, and TAQMAN ® probe detection systems. Each sample was amplified in a separate PCR reaction using the same type of primers. 3.1 Primer-dimer analysis
  • primer-dimer amplification data (hereinafter "pd data") was analyzed for eventual use in accounting for the effects of competition between primer-dimer amplification and lambda DNA amplification.
  • Primer-dimers a non-specific material, were amplified in the absence of the target DNA.
  • the pd data includes data from two samples run in individual experiments. Forty PCR cycles were conducted with primers present, but without any Lambda DNA present, in the PCR tube. Primer-dimer formation was monitored cycle-by-cycle by detecting fluorescence of SYBR GREEN I ® .
  • FIG. 2A depicts raw pd data from the two samples.
  • FIG. 2B depicts the pd data after the data has been cut and scaled.
  • the first four cycles of the pd data were removed and the cycles renumbered to start from zero.
  • the raw pd data was scaled so that the highest curve has a maximum value of 1.0. All other curves were scaled proportionally to this curve.
  • scaling factors were calculated using the first derivative. Curves that have not started growing yet were not scaled up, whereas curves that had reached plateau were scaled to 1. Curves in between were scaled up proportionally.
  • pd samples Primer-dimer samples that did not have any growth over the amplification cycles were removed from the pd data.
  • the second derivative of each pd sample curve was used to determine whether the pd sample had growth or not. Pd samples having a second derivative less than 1 were considered to not have any growth and were removed from the scaled pd data.
  • Fake data was added to the raw pd data to test the effectiveness of the data scaling.
  • the linear line in FIG. 2A depicts the fake data used to test the data scaling.
  • the range of the fake data was set equal to the range of the raw pd data.
  • FIG. 2B depicts that the fake data was effectively scaled to zero.
  • target data rough scaling of target nucleic acid data
  • detection system specific scaling was conducted.
  • a final processing step was performed that was the same for all detection systems.
  • Raw SYBR GREEN I ® target data is depicted in the upper half of FIG. 3A.
  • the background noise was then subtracted.
  • the SYBR GREEN I ® detection system it was assumed that the - background noise was zero, because the detection signal is usually very strong.
  • the background noise was the average of the negative samples.
  • the cut target data was shifted to start from 0.
  • the cut target data was scaled so that the maximum value for the highest curve is 1 and all of the other curves were scaled proportionally to this curve. Each curve was scaled to start at 0.
  • the cut SYBR GREEN I ® target data is depicted in the lower half of FIG. 3 A.
  • FIG. 3B depicts fit SYBR. GREEN 1 ® target data ("fit”). The first derivative of the fit target data was then calculated.
  • FIG. 3C depicts the first derivative of the fit SYBR GREEN I ® target data ("fltd”). Next, the second derivative was taken.
  • FIG. 3D depicts the second derivative of the fit SYBR GREEN I ® target data ("fitdd”). Next, three functions were generated - phi, phid, and phidd. Phi is the polynomial basis for the quintic splines of the scaled target data and is illustrated in FIG.
  • the sample did not show target nucleic acid growth.
  • the deviation of each line from the fit target data was also calculated (see, e.g., FIG. 4E for SYBR GREEN I ® target data). If the deviation was ⁇ 0.001, then the curve was considered not to show template growth.
  • the fit target data was scaled using the deviations from the baseline as a scaling factor. The fit target data was scaled between 0 and 1 depending on the stage (early, middle, late of amplification reaction).
  • FIG. 4F depicts the fit target data after scaling and filtering for SYBR GREEN I ® target data. 3.2.2 Detection System Specific Scaling
  • Rough scaled SYBR GREEN I ® target data was scaled using the maximum value of the first derivative fitd (see, e.g., FIG. 3C).
  • the first derivate of the last cycle was compared to the cycle where the first derivative was maximal.
  • the function bt was used to scale the target data with regard to the degree of completion of the amplification reaction. The behavior of bt and scale is illustrated in FIG. 5. If at the final cycle the curve had not yet started to grow, the data was not scaled up. If at the final cycle the curve had reached plateau, the max value was set to one. Otherwise, the data was scaled up proportionally. The scaling was used to generate a first guess for later scaling processes.
  • FIGS. 6A and 6B illustrate hybridization probe raw data and hybridization probe rough scaled data, respectively. The following was used to determine the scaling factor between fluorescence signal and copy number:
  • FIG. 6C illustrates X n after the first turn of the loop.
  • FIG. 6D illustrates the derivative of x n with respect to d n after the first loop.
  • FIG. 6E illustrates the derivative of X n with respect to s after the first loop.
  • FIG. 6F illustrates the calculated relationship between d n and x n .
  • G is the quotient of two polynomials: g n /g d , where g n is a second degree polynomial and gd is a zero degree polynomial. Therefore, gd was set to 1, which means G equals g n .
  • the scaling factor "s" was kept constant while the coefficients of g n were being optimized. Therefore,
  • x(j+l) x(J) + X(J)-O - S-X(J))* gn / gd (41)
  • dgnP is the derivative of gn with respect to P, which equals (1 - Xi) in this case
  • dgnx is the derivative of g n with respect to x,.
  • the estimated growth for each target DNA sample was calculated for each cycle, using the previously calculated relative initial quantity of target DNA at cycle 0 as the starting point.
  • the relative copy number i.e., relative number of target DNA copies
  • the result for target DNA was stored in the variable z and the result for the primer-dimers was stored in the variable zp, both illustrated in FIG. 7.
  • the fit of the calculated z data to the x n data was then calculated.
  • estimated growth curves were calculated for the primer-dimer data depicted in FIG. 2A.
  • estimated growth curves were calculated for the raw data depicted in the upper panel of FIG. 3 A.
  • the estimated growth curves are illustrated in FIGS. 8A and 8B, respectively. Initial quantities for the estimated growth curves were calculated using bisection.
  • primer-dimer growth (yj) and target DNA growth (xj) in each cycle were then calculated using equations (39), (40), (45), and (46). The total growth was contained in variable z.
  • Pj 1-zj-xj.
  • Pj 1-yj-xj.
  • FIG. 8C depicts the estimated template ("target DNA”) growth, estimated primer-dimer growth, estimated template efficiency, and estimated primer-dimer efficiency for the hybridization probe data.
  • Table 1 lists the calculated initial quantities of target DNA per sample. Before optimization is the calculated initial quantity prior to taking into account the effects of competition. After optimization is the calculated initial quantity after such calculations.
  • Lambda DNA was amplified using a RTD-PCR Roche Applied Science Lightcycler ® . Amplifications were conducted using SYBR GREEN I ® , hybridization probe, and TAQMAN ® probe detection systems.
  • This example is similar to Example 3.
  • a SYBR GREEN I ® positive sample was added as a reference for the initial raw scaling of primer-dimer negative samples, when a detection system other than SYBR GREEN I ® was used.
  • the primer-dimer analysis was similar to that of Example 3. Fake data was added to the raw pd data to test the effectiveness of the data scaling.
  • the range of the fake data was changed for detection systems other than SYBR GREEN I ® . The range was set equal to the range of the SYBR GREEN I positive samples that were included in the experiment setup.
  • FIG. 9A illustrates a calibration curve generated using SYBR GREEN I ® target data and iterative growth functions.
  • FIG. 9B illustrates estimated target DNA growth and efficiency using the SYBR GREEN I ® detection system and a competition model.
  • FIG. 9C illustrates the relative initial quantities calculated with primer-dimer correction that were used to generate the calibration curve of FIG. 9A.
  • FIG. 9C also illustrates the relative initial quantities calculated without primer-dimer correction ⁇ i.e., without accounting for the effects of primer-dimer competition).
  • FIG. 1OA illustrates a calibration curve generated using the prior art second derivative maximum method and the same SYBR GREEN I ® data used to generate the calibration curve of FIG. 9A.
  • FIG. 1OB illustrates quantification verification of the second derivative maximum method data.
  • Certain embodiments of the invention maybe used to generate calibration curves that are more precise at lower target nucleic acid concentrations than with prior art analysis methods. For example, with the prior art second derivative maximum method of the SYBR GREEN I ® data depicted in FIG. 1OA, it would be difficult to distinguish between initial samples sizes that are 10 5 or smaller. In contrast, using the same data, a calibration curve generated using embodiments of the invention allows for initial sample sizes to be distinguishable to a much lower range.
  • the upper left panel of FIG. 1 IA illustrates the raw data illustrated in FIG. 6A generated using a hybridization probe detection system.
  • the upper right panel illustrates the scaled data illustrated in FIG. 6B.
  • the bottom panel illustrates raw SYBR GREEN I ® negative data (i.e., raw primer-dimer data).
  • FIG. 11 B illustrates estimated target DNA growth and efficiency and estimated primer-dimer growth and efficiency using iteratively fit growth functions.
  • the upper right panel of FIG. 11C illustrates a calibration curve generated using iterative growth functions fit to the hybridization probe data.
  • the upper left panel of FIG. 11C illustrates the calculated initial quantities that were used to generate the calibration curve.
  • the bottom panels illustrate quantification verification and results using iteratively fit growth functions.
  • FIG. 12 illustrates a calibration curve generated using the raw hybridization probe data illustrated in FIG. 1 IA and the second derivative maximum method.
  • FIG. 12 also illustrates quantification verification and results using the second derivative maximum method.
  • FIGS. 13-16 illustrate the use of internal controls during target DNA amplification.
  • FIG. 13 illustrates use of the direct method.
  • FIG. 13 in the upper left panel, illustrates raw fluorescence data from RTD-PCR of lambda DNA ("target DNA") with an internal control. TAQMAN ® hydrolysis probes were used for detecting target DNA and internal control amplification in a two color experiment. The target DNA signal was at a distinguishable wavelength from the internal control signal.
  • the upper right panel illustrates just the fluorescence data from the target DNA growth.
  • FIG. 13 also illustrates calculated target DNA efficiency and internal control efficiency.
  • FIG. 14 illustrates a calibration curve generated using the data illustrated in FIG. 13 and the direct method.
  • FIG. 14 also illustrates the data used to generate the calibration curve and quantification verification and results based on that data.
  • FIG. 15 illustrates a comparison between the direct method and the indirect method using the data illustrated in FIG. 13.
  • the dashed lines are theoretical curves calculated with embodiments of the invention.
  • the solid lines represent the raw data collected.
  • FIG. 16 illustrates a calibration curve generated using the indirect method.
  • FIG. 16 also illustrates the data used to generate the calibration curve and quantification verification and results based on that data.

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Abstract

L'invention concerne un procédé de quantification de séquences d'acides nucléiques dans des réactions d'amplification ou de transcription inverse utilisant un système d'amplification en chaîne par polymérase (PCR) à détection en temps réel (RTD). L'invention comprend la formulation de modèles mathématiques à partir de descriptions cinétiques de PCR pour différents systèmes RTD; et le développement d'algorithmes d'application pour ces modèles en vue d'analyser quantitativement des expériences RTD-PCR. L'invention comprend également une analyse quantitative des effets de l'amplification non spécifique (réactions secondaires) sur le rendement d'une amplification cible spécifique. L'invention comprend enfin l'analyse quantitative de multiples amplifications simultanées spécifiques (réactions multiplexes) comprenant des expériences de quantification relatives témoins internes.
PCT/US2008/067496 2007-06-19 2008-06-19 Procédés d'analyse d'amplification d'acides nucléiques WO2008157689A2 (fr)

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US10482995B2 (en) 2013-05-15 2019-11-19 Thorne Diagnostics, Inc. Nucleic acid amplification signal acquisition and signal analysis

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