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WO2006037207A1 - Appareil, processus et methodes destines a etre utilises dans la pcr quantitative - Google Patents

Appareil, processus et methodes destines a etre utilises dans la pcr quantitative Download PDF

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WO2006037207A1
WO2006037207A1 PCT/CA2005/001354 CA2005001354W WO2006037207A1 WO 2006037207 A1 WO2006037207 A1 WO 2006037207A1 CA 2005001354 W CA2005001354 W CA 2005001354W WO 2006037207 A1 WO2006037207 A1 WO 2006037207A1
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signal
fluorescence
reaction
amplification
nucleic acid
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Robert G. Rutledge
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National Research Council Of Canada
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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
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/695—Preprocessing, e.g. image segmentation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F2218/00—Aspects of pattern recognition specially adapted for signal processing
    • G06F2218/08—Feature extraction
    • G06F2218/10—Feature extraction by analysing the shape of a waveform, e.g. extracting parameters relating to peaks

Definitions

  • the invention relates to the field of nucleic acid quantification, particularly as it relates to the polymerase chain reaction ( 11 PCR").
  • PCR polymerase chain reaction
  • thermocycling reaction temperature is repetitively cycled (called thermocycling) for a sufficient number of times to amplify the target to saturating amounts (generally 50 thermocycles).
  • fluorescence intensity is related to the mass of amplified DNA. Fluorescent detection of amplified DNA not only allowed the kinetics of PCR amplification to be determined with high resolution, it also allowed automation via software data analysis.
  • PCR is the basis for a number of new biomedical diagnostic applications, in addition to quantitative applications for molecular genetics and genomics research (e.g. gene expression analysis, pathogen detection, genetic analysis, among many others).
  • the inventors of this approach (Higuchi et al. 1993; EP0640828) defined this point by selecting a threshold fluorescence, from which a fractional threshold cycle (Ct; also called the crossing point or CP) is derived for each amplification reaction.
  • Ct fractional threshold cycle
  • Rutledge and Cote (2003), which describes the underlying mathematics and the application of standard curves for converting Ct values into the number of target molecules.
  • the threshold approach has historically dominated the field of quantitative PCR. Although several variants have appeared which attempt to automate the selection of the fluorescence threshold, e.g. based upon maximum or minimum values of the first and/or second derivatives of the reaction fluorescence curve (Wittwer, US 6.303.305 EM ; McMillan, US 6,713,297 B2), none of these modify how target quantification is accomplished.
  • the threshold approach as currently practiced has several weaknesses, directly relating to two aspects encompassing amplification efficiency. First is that amplification efficiency must be known in order to achieve target quantification.
  • Amplification efficiencies in turn can only be determined accurately via construction of standard curves (US 2002/0058262 A1 , US 6,691 ,041 B2), which are difficult to construction, prone to errors and are impractical for high-throughput applications.
  • PCR are not fully satisfactory.
  • the "threshold-based method" that has dominated quantitative application of real time PCR to date can give rise to difficulties in implementation, insufficient reliability, and difficulties with quality control.
  • the necessary assumption that amplification efficiency of samples is equal to that of the DNA standards used for absolute quantification has been shown to be invalid in many applications, resulting in large and unpredictable errors. Occurrences of such errors are of paramount importance for many diagnostic applications that require a high level of quantitative accuracy and reliability, such as for determination of viral load or residual disease.
  • the innate limitations of the threshold-based methods have thus precluded broad implementation of quantitative kinetic PCR in many types of biomedical and diagnostic applications.
  • target polynucleic acid sequences including DNA and RNA sequences (called the "target") contained within a sample.
  • This is accomplished by linear regression analysis of data generated during PCR amplification (using standard, commercially available inst ⁇ i mentation and enzyme kit), from which values for two parameters are derived. These are then inputted into a novel mathematical function from which target quantity is calculated.
  • the final step is conversion of quantitative units to the number of target molecules, based upon correlation of reaction fluorescence to DNA mass, a process called herein "optical calibration”.
  • the present invention greatly simplifies target quantification by circumventing the need for curve fitting, using instead an easy to implement approach based upon linear regression analysis. Combined with methods for establishing and monitoring the accuracy of quantitative scale via optical calibration, the present invention not only permits conversion of fluorescence units Into the number of target molecules, but as well provides effective quality control and assurance over quantitative scale. This enables unprecedented capabilities for verification of quantitative accuracy, and for development of generally applicable quantitative standards for calibrating real time PCR thermocyclers.
  • FIGURE 1 A is a graphical depiction of an embodiment of sigmoidal functions describing amplicon accumulation and cycle efficiency.
  • FIGURE 1 B is a graphical depiction of an example of cycle efficiency plotted against reaction florescence.
  • FIGURE 2A is a graphical depiction of an example of loss in amplicon efficiency in relation to DNA accumulation.
  • FIGURE 2B is a graphical depiction of an example of fluorescence readings
  • FIGURE 2C is a graphical depiction of an example of ratio-derived amplification efficiency plotted against cycle number.
  • FIGURE 3A is a graphical depiction of an example of amplification profiles of reaction fluorescence vs. cycle number with varying reaction volume.
  • FIGURE 3B is a graphical depiction of an example of ratio-derived cycle efficiency vs. reaction fluorescence with varying reaction volume.
  • FIGURE 4A is a graphical depiction of an example of amplification profiles of reaction fluorescence vs. cycle number with varying annealing and elongation times.
  • FIGURE 4B is a graphical depiction of an example of ratio-derived cycle efficiency vs. reaction fluorescence with varying annealing and elongation times.
  • FIGURE 5 is a graphical depiction of an example of drifting plateau produced by low base amplification efficiencies.
  • FIGURE 6 is a graphical depiction of an example of the relationship between fluorescence and DNA mass.
  • FIGURE 7 is a graphical depiction of mathematical modeling of PCR amplification.
  • FIGURE 8 is a depiction of a screen shot of an MS Excel TM worksheet used in an example of LRE quantification.
  • this aspect of the invention also permits effective monitoring accuracy-of-scale, in addition to greatly facilitating high-throughput applications, in part by circumventing the need to prepare quantitative standards for each individual target, as is required using current threshold- based methodologies.
  • PCR quantification via LRE is an eight-step process, although certain steps may be useful alone or in sub-combinations.
  • the process can be conducted in eight steps, for example:
  • reaction fluorescence is assayed at a selected temperature such that reaction fluorescence is directly proportional to the amount of DNA present in the reaction; reaction fluorescence is measured following each thermocycle, producing an profile of the DNA amplification process (e.g. Figure 2B)
  • Amplification efficiency is calculated for each thermocycle from the ratio of reaction fluorescence (FQ) to the reaction fluorescence from the previous thermocyde (Fc-i), generating what is called the ratio-based estimate of amplification efficiency (ER):
  • E R is plotted against reaction fluorescence (F c ). producing a line from which the Y intercept and slope are determined using a standard linear regression analysis ( Figure 2). These represent two key kinetic parameters that define the shape of the amplification curve, called maximal amplification efficiency (E max ) and rate-of-decay of amplification efficiency ( ⁇ E) as described by:
  • the maximal fluorescence (F ma ⁇ ) produced by the amplification is in turn determined by the ratio of E ma ⁇ to its rate-of-decay ( ⁇ E):
  • Equation 2 can also be used to predict the efficiency of each cycle (Ec) based upon its corresponding fluorescence (F c ), which enables quality assessment of the fluorescence dataset based upon the level of correlation between ER with E c :
  • Er ⁇ x and F max are placed into a novel mathematical function (equation 5) from which target quantity is calculated using cycle number (C) and the corresponding reaction fluorescence reading (F c ). This produces a target quantity in the fluorescence i units (F 0 ), calculated for each thermocycle (within the amplification curve), from which an average and standard deviation of target quantity is calculated:
  • a related sigmoid function can be used to predict reaction fluorescence (F P ) once Fo is calculated. This enables assessment of the quality of the fluorescence dataset based upon the level of correlation between Fp and F c :
  • Target quantity is converted into DNA mass (Mo) by multiplication with a calibration factor (CF) that relates reaction fluorescence units (FU) to DNA mass (nanogram) and is expressed in units of nanograms/FU.
  • CF calibration factor
  • Amplification efficacy can be assessed for individual amplification reactions via maximal amplification efficiency; this enables presence of PCR inhibitors or ineffective reaction preparation to be assessed for each individual amplification reaction
  • Target quantification can be accomplished from data obtained from a single amplification reaction; threshold-based quantification can only be accomplished by comparison to other samples, with absolute quantification (i.e. the number of target molecules) requiring PCR amplification of DNA standards in which the quantity of target is known 5.
  • absolute quantification i.e. the number of target molecules
  • An alternative approach encompasses a process called "optical calibration" that exploits the discovery that quantitative scale is directly linked to am pi icon fluorescence through its relationship to DNA mass, such that target quantification can be achieved by correlating reaction fluorescence to the DNA mass present in the reaction mixture.
  • Optical calibration is important in some applications due to the uncalibrated nature of fluorescence units produced by real time PCR 1 such that fluorescence units generated by each individual thermocycler is unique to both the optical characteristics of its instrumentation and the optical characteristics of the reaction mixture. Thus it is generally important to derive a conversion factor for each thermocycler and reaction configuration. DNA mass can be accurately determined based upon the reaction fluorescence as disclosed herein. This in turn enables the number of target molecules to be calculated, without having to resort to a PCR-generated standard curve.
  • optical calibration as described herein enables a higher level of quality control and assurance of quantitative scale, than attainable using current methodologies. Furthermore, it enables development of universal standards that could facilitate development of worldwide standards for establishment and verification of quantitative scale, a deficiency that currently plagues quantitative PCR (Bustin 2002).
  • LRE quantification generates target quantity in fluorescence units (Equation 5), which although useful for comparing the relative quantities of target in different samples, is in some applications less useful than quantification in units of target molecules. This is similar to the deficiency of quantification via curve-fitting as described by Liu and Saint (2002) that also produces target quantity in fluorescence units. Conversion of fluorescence units into the number of target molecules substantially Increases the utility of the LRE-based quantification, particularly for biomedical diagnostics in which accurate, verifiable quantification of the number of target molecules is paramount (e.g. for viral load and residual disease).
  • Optical calibration is a multi-faceted process in which fluorescence units are correlated to DNA mass through derivation of a calibration factor (CF).
  • CF calibration factor
  • optical calibration enables assessment of optical precision and accuracy of real-time instruments in an effective, accurate and timely manor.
  • verification of accuracy-of-scale can be accomplished by comparing optical calibrations obtained by two independent methods, a capability lacking under currently applied methodologies.
  • the nucleic acid will be DNA. In some cases it will be
  • the nucleic acid standard is preferably DNA when the target is DNA and RNA when the target is RNA.
  • DNA from the bacteria phage lambda is an effective standard for optical calibration in many cases.
  • a quantified stock of the nucleic acid standard is prepared, from which diluted samples are made.
  • Optical calibration encompasses two distinct methods that are linked through the use of the same quantified stock of the nucleic acid standard.
  • the first method uses PCR amplification of a target(s) within the nucleic acid standard, using a diluted sample containing an appropriate amount of the nucleic acid standard.
  • the nucleic acid mass of the target (M 0 ) is determined by first calculating the number of nucleic acid molecules of the standard contained in the sample (No):
  • N 0 (S M x9.1x10 11 yS s Equation 9
  • S M is the mass of the nucleic acid standard in nanograms and Ss is the molecular size in base pairs (where the standard is DNA) of the nucleic acid standard.
  • Equations 9 and 10 can be combined and simplified:
  • the LRE process is used to determine the target quantity in fluorescence units (Fo) and the conversion factor (CF 0 ) determined :
  • CFo is the number nanograms per fluorescence unit (ng/FU) based upon real time PCR quantification of the nucleic acid standard.
  • the second method is called "direct calibration" and does not involve PCR amplification.
  • Direct calibration can be conducted by preparing mock PCR reaction mixes identical to that used for PCR amplification, except that they contain the nucleic acid standard in an amount(s) similar to that generated within a typical amplification profile (approximately 10-50 ng for a 25 ⁇ l reaction volume). Reaction mixes lacking the nucleic acid standard are also prepared for determining background fluorescence (F b ). Note also that is ) not necessary to use the same standard as that used for PCR-ba ⁇ ed calibration, such that any source of nucleic acid can be used assuming that it is accurately quantified.
  • the mock reaction mixes are placed into the real time thermocycler and the block temperate increased to the same temperature used to read reaction fluorescence during PCR amplification. This is necessary because reaction fluorescence is temperature dependent. Once that the selected temperature is reached, fluorescence reading(s) are taken (Fd).
  • CF d is the "direct" calibration factor in units of ng/FU
  • S M is the mass of the nucleic acid standard in nanograms
  • Fo is the fluorescence of the mock PCR reaction at the read temperature
  • F b is the background fluorescence at the read temperature produced by a mock PCR reaction containing no DNA standard.
  • optical calibration In addition to enabling the conversion of Fo into No 1 optical calibration can also be used to evaluate optical precision, that in turn has a direct impact on quantitative accuracy.
  • the most fundamental approach is assessment of the correlation between CF d and CF 0 ; that is quantitative calibrations derived by two independent methods who's level of correlation provides an indication of the precision of the optical calibration process.
  • the level of optical stability over time can be evaluated through the correlation between multiple applications of the optical calibration process, conducted over time. This in turn provides an indication of how frequently a real time thermocycler needs be recalibrated in order to maintain a specified tolerance for accuracy- of-scale, and thus ultimately quantitative accuracy.
  • optical calibration allows one or more specific advantages, such as: 1. Direct calibration enables optical calibration without PCR amplification; effective, easy to execute, and reliable 2. Enables verification of optical calibration by assessing the correlation between amplification of a quantified standard (CFo) with that of direct calibration (CF d ) 3. Provides a single quantitative scale for all targets; prevailing threshold-based methods requires preparation of quantified standard for each Individual target; this process eliminates this limitation through the application of a single DNA standard, greatly facilitating high throughput applications 4. Use of an easily accessible DNA standard facilitates standardization of optical calibration and thus for establishment of quantitative scale, a process that is also platform independent (i.e. not unique to any individual real time thermocycler machine, model or manufacture) 5. Direct calibration can be verified via application of multiple DNA sources; that Is direct calibration is not limited to a single source of
  • DNA standard 6 Verification of PCR-based optical calibration through amplification of multiple targets within the DNA standard; that is to conduct optical calibrations via amplification of multiple targets using the same DNA standard preparation 7. Verification of PCR-based calibration via direct calibration using the same DNA standard preparation; this provides a direct link between the two calibrations methods providing further verification of the accuracy of quantitative scale 8. Enables standardization by adoption of specified DNA sources for preparation of standards for optical calibration, based upon several desirable attributes such as accessibility, purity, known size and sequence
  • a method for identifying a patient having a condition of interest involving or identifiable by the abundance of a least one selected nucleic acid sequence comprising:
  • f) determine the Y intercept and slope of the line from step e, where in the intercept is the maximal amplification efficiency (E m * ⁇ ) and the slope is the rate-of-decay of the base amplification efficiency ( ⁇ E); g) determining F max wherein
  • CF is determined using a process called optical calibration, in which reaction fluorescence is correlated to polynucleic acid mass j) calculating the number of polynucleic acid molecules based upon polynucleic acid mass of the target (Mo) based upon the size of the amplified region (A s ), wherein
  • k comparing the number of polynucleic acid molecules calculated above to a predetermined number or range of polynuleic acid molecules associated with a condition of interest.
  • nucleic acid quantity can be used to assess the abundance of a nucleic acid sequence of interest from a sample, for instance to determine if it exceeds a threshold level which is considered "safe” or "normal” for the healthy population.
  • a machine readable media such as a read-only memory, a random access memory, an optical or magnetic disk or tape, or other device for storage of information, containing instructions for carrying out one or more of the processes listed above.
  • Such media can be used together with a polymerase chain reaction apparatus to assess PCR results on an ongoing basis, or may be used on a separate processor to examine cycle-specific signal data obtained from a PCR apparatus.
  • the "signal" to be assessed relating to nucleic acid incorporation may be of any suitable type. At present, fluorescence is a popular signal method used to track PCR reactions. However, In the past radio-labeled nucleotide incorporation was used, and such methods may still be preferred for particular applications.
  • Figure 1 depicts sigmoidal modeling of the polymerase chain reaction reveals symmetry between amplicon DNA accumulation and loss in amplification rate.
  • A Plots of the sigmoid functions describing amplicon accumulation (equation 1; solid line) and cycle efficiency (equation 2; dashed line) illustrate a symmetrical relationship, where Cy 2 defines the fractional cycle when both reach precisely half of their respective maxima.
  • F 0 is target quantity expressed in fluorescence units
  • E maX is the maximal amplification efficiency
  • F ma ⁇ is the maximal reaction fluorescence.
  • B Plotting cycle efficiency against reaction fluorescence produces a line, indicating that the progressive loss in cycle efficiency is directly related to amplicon accumulation.
  • Figure 3 Reaction volume is a determinant of F max .
  • A Amplification profiles of reactions containing an identical quantity of target but of increasingly greater volume (20 ⁇ l (•), 30 ⁇ l (T), 40 ⁇ l ( ⁇ ), 50 ⁇ l (A) and 60 ⁇ l ( ⁇ )).
  • B Ratio-derived estimates of cycle efficiency (E Cl equation 3) were determined from the fluorescence readings generated by each amplification reaction and plotted against the respective reaction fluorescence (F c ), producing a linear representation of each of the amplification profiles shown in (A) (Fig. 1B).
  • Figure 4 Time of annealing and elongation is a determinant of maximal amplification efficiency.
  • A Amplification profiles of reactions containing identical quantity of target but with different annealing and elongation (A&E) times (4 min (T) 1 2 mi ⁇ (A) 1 1 min ( ⁇ ) and 0.5 mi ⁇ (•)). This reveals that both maximal reaction fluorescence and slope of the amplification profile decrease with decreasing A&E time, along with a progressive shift in profile position consistent with a reduced maximal amplification efficiency.
  • B LRE plots show that reduced time of A&E produces a decreased maximal amplification efficiency (Y intercept, E max ), but has minimal, if any impact on the rate-of-loss in cycle efficiency (slope, ⁇ E). Table IV relates to figure 4 and provides a summary of the linear regression analysis (LRE analysis; equation 4) of the plots shown in (B). Vol., reaction volume; r 2 , linear correlation coefficient.
  • Figure 6 Optical calibration via two distinct processes.
  • a quantified, lambda DNA stock solution was employed as a standard for optical calibration using both direct (A and B) and PCR-based (C) determination of the calibration factor (CFd and CFo respectively) used for converting fluorescence units into DNA mass.
  • a linear relationship of DNA mass with fluorescence is found over the range of 15-50 ⁇ g of lambda DNA when placed Into mock PCR reactions (25 ⁇ l containing all of the components in a standard amplification reaction).
  • the tubes were placed into the thermocycler, heated to the read temperature used for real time PCR (68 0 C) and multiple optical reads taken. Following subtraction of the fluorescence produced in the absence of lambda DNA (i.e.
  • the predicted target quantity in DNA mass was calculated using equation 11 based upon the known size of the lambda DNA molecule (48,502 bp; S 5 ) and the amplicon size of each target (As). Following four replicate amplification for each target was conducted using the same thermocycling conditions and read temperature (68 0 C), LRE was used to determine Fo (target quantity in fluorescence units) from which a calibration factor (CF 0 ) was calculated for each target and an average taken. Both methods produced an average CF that agree within ⁇ 4%, providing verification of accuracy, at least based upon a shared DNA standard. Fluor., fluorescence.
  • Figure 7 Mathematical modeling of PCR amplification and assessment of the quantitative precision provided by LRE analysis.
  • A Replicate amplifications were conducted on ten fold dilutions of lambda genomic DNA and LRE quantification conducted on each amplification profile. Actual (•) and predicted (o, equation 7) reaction fluorescence for each target quantity are plotted against cycle number.
  • the table below depicts a summary of the LRE analysis conducted on the fluorescence profiles presented in Figure 7A, along with the calculated target quantity (F 0 , equation 6. Table II).
  • R 2 nonlinear correlation coefficient (see Materials and Methods and related Figures)
  • FIG. 8 Screen shot of the MS Excel worksheet used for the LRE quantification conducted on the 18,800 molecule (1 pg) lambda genome amplification presented in Fig. 7. Graphs of the LRE analysis and comparison of actual to predicted fluorescence profiles are presented, with arrows denoting the range of cycles included in the analysis. Following background subtraction, reaction fluorescence readings from four replicate reactions were averaged (F 0 ), imported into MS Excel, and ratio-derived estimate of cycle efficiency (Ec) calculated using equation 1.
  • Ec is the amplification efficiency at cycle C, also referred to as "cycle efficiency".
  • cycle efficiency amplification efficiency decreases continuously such that each cycle has a unique amplification efficiency.
  • the principle of this and other insights can be illustrated by plots of these equations, the most notable being a striking symmetry between amplicon DNA accumulation and loss in amplification efficiency ( Figure 1).
  • Equation 4 allows determination of cycle efficiency based solely upon reaction fluorescence.
  • equation 14 can be modified by first substituting for k using equation 17, which simplifies to:
  • equation 19 substituting for C 1 / 2 and simplifying, equation 19 becomes:
  • equation 5 allows target quantity to be determined directly from reaction fluorescence, once estimates for E B and ⁇ E are obtained via linear regression using equation 2, a process termed "linear regression of efficiency" or LRE.
  • LRE linear regression of efficiency
  • mechanistic modeling can also have important practical applications, as for example, in the evaluation and optimization of amplification efficacy. These encompass issues familiarity to most, but which to date have received little or no experimental support. With respect to reaction preparation, examples include the relative impact of primer and enzyme concentration, or of amplicon size on quantitative efficacy. Of possibly greater importance is the ability to define in analytical terms, the impact of thermocycling parameters. As demonstrated here, the time of annealing and elongation has a major influence on amplification efficiency.
  • LRE will have a major impact on extending the quantitative capabilities and applications. Indeed, all of the quantitative attributes of sigmoidal curve-fitting using Boltzmann-based sigmoid functions, are directly applicable to an LRE-based approach. These include the ability to monitor amplification efficacy within individual reactions, and to conduct quantification without having to construct standard curves. A major advantage of LRE is however, circumventing the complexities of nonlinear regression (i.e. curve fitting), and in particular, the difficulties produced by a drifting plateau..
  • This protocol preferably incorporates a two-step cycling regime consisting of a denaturation step at or about 95 0 C for 10 seconds, and a combined annealing and elongation (A&E) step at 65 0 C for 180 seconds.
  • A&E combined annealing and elongation
  • the selection of temperature and time of the A&E step influences amplification specificity and high amplification efficiency. It is within the skil of one in the art, in light of the disclosure herein, to select suitable annealing and elongation temperatures.
  • Typical temperature ranges are 60 to 75 degrees Celsius, sometimes 65 to 70 degree Celsius. (These temperature ranges are driven significantly by the activity and stability of the Taq enzyme. One skilled in the art in possession of a different polymerase would be able to select a suitable temperature in light of the disclosure herein.)
  • LRE analysis was further utilized to examine the efficacy of primer design methodologies. To date, it is common practice to utilize specialized software programs to design primers, based upon the objective of reducing the frequency of non-specific amplification products and promoting the general effectiveness of primer pair combinations. Despite the" general perception that careful selection of the DNA sequence encompassed by a primer is required for a high rate of success, LRE analysis, in combination with the cycle regime described above, led to the discovery that only a single parameter is important for effective primer design. This parameter is primer melting temperature (Tm), a parameter determined solely by primer length and base pair composition; the actual DNA sequence within a primer was generally found to have no substantive impact on amplification efficiency, nor on the determination of amplification specificity.
  • Tm primer melting temperature
  • this discovery greatly facilitates large-scale application of qPCR by allowing the position of the primer within the target to be selected without restriction. In practice this involves selecting a 3 prime starting position, generally near to the stop codon for mRNA quantification, and to increase the length of the primer until the calculated Tm reaches 70 0 C. Combined with the universal cycling regime described above, LRE analysis of a large number of primer pairs has demonstrated that >95% of all primer pairs produce high amplification specificity, with all primer pairs producing amplification efficiencies within the range of 95-100% as determined by LRE analysis.
  • Nonlinear correlation coefficients of predicted fluorescence profiles presented in Fig. 7 were calculated over the range of cycles included in the
  • Fc reaction fluorescence and F P is the predicted reaction fluorescence at cycle C, with F ⁇ being the average reaction fluorescence over the range of cycles used in the LRE analysis.
  • Bacteriophage lambda DNA was used as template for all amplification reactions, which were conducted with a MX3000P spectrofluorometric thermal cycler (Stratagene) using QuantiTectTM SYBR ® Green PCR Kit (Qiagen Inc.). Data presented in Fig. 3 and 4 were generated using primer pair Lam K7-K8 (amplicon size 225 bp), and primer pair Lam K7-K12 (amplicon size 150 bp) was used to generate the data presented in Fig. 7.
  • Lam K7 CTGCTGGCCGGAACTAATGAATTTATTGGT Lam K8: ACCGAGTTCAGAAATAAATAACGCGTCGCCGGAA
  • Lam K12 ATGCCACG ATG CCTCATCACTGTTG Unless otherwise indicated, four replicate amplification reactions containing 10 pg lambda DNA and 500 ⁇ M of each primer were conducted, v starting with a 15 min incubation at 95 0 C, followed by a cycling regime of 95 0 C-10 sec, 65 °C-180 sec, and reaction fluorescence determined by averaging five fluorescent readings taken at the end of each cycle. Each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primer dimers. Following fluorescence background subtraction, fluorescence readings from the four replicates were averaged and exported into MS Excel for analysis.

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Abstract

L'invention porte sur un appareil, sur un support lisible par machine, sur des méthodes et des processus permettant d'effectuer une amplification en chaîne par polymérase quantitative, en temps réel (PCR) et quantifier une séquence polynucléotide cible unique contenue dans un échantillon. Pour cela, on procède à une analyse de régression linéaire des données générées pendant l'amplification PCR d'où sont dérivées des valeurs provenant de deux paramètres: le rendement maximal de l'amplification et le taux de décroissance du rendement de l'amplification. On introduit ensuite ces valeurs dans une fonction mathématique à partir de laquelle on calcule la fluorescence maximale, la quantité cible ou la quantité d'acide nucléique amplifié à un cycle spécifique. L'étape finale est la conversion des unités quantitatives en nombre de molécules cibles en fonction de la corrélation de la fluorescence de la réaction par rapport à la masse d'ADN. On identifie ainsi un patient présentant un état entraînant l'abondance d'une séquence d'acides nucléiques sélectionnée en comparant le nombre calculé avec un nombre prédéterminé de molécules d'acide polynucléique.
PCT/CA2005/001354 2004-10-05 2005-09-06 Appareil, processus et methodes destines a etre utilises dans la pcr quantitative WO2006037207A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8346485B2 (en) 2008-11-25 2013-01-01 Quest Diagnostics Investments Incorporated Methods and apparatuses for estimating initial target nucleic acid concentration in a sample by modeling background signal and cycle-dependent amplification efficiency of a polymerase chain reaction
US8700381B2 (en) 2009-04-16 2014-04-15 Koninklijke Philips N.V. Methods for nucleic acid quantification
EP2238262A4 (fr) * 2007-12-28 2015-04-22 Abbott Lab Analyse de discrimination allélique utilisant une valeur apparentée à l'efficacité (efr)
CN116092585A (zh) * 2023-01-30 2023-05-09 上海睿璟生物科技有限公司 基于机器学习的多重pcr扩增优化方法、系统、设备及介质

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EP2238262A4 (fr) * 2007-12-28 2015-04-22 Abbott Lab Analyse de discrimination allélique utilisant une valeur apparentée à l'efficacité (efr)
US8346485B2 (en) 2008-11-25 2013-01-01 Quest Diagnostics Investments Incorporated Methods and apparatuses for estimating initial target nucleic acid concentration in a sample by modeling background signal and cycle-dependent amplification efficiency of a polymerase chain reaction
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CN116092585A (zh) * 2023-01-30 2023-05-09 上海睿璟生物科技有限公司 基于机器学习的多重pcr扩增优化方法、系统、设备及介质
CN116092585B (zh) * 2023-01-30 2024-04-19 上海睿璟生物科技有限公司 基于机器学习的多重pcr扩增优化方法、系统、设备及介质

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