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WO2011008818A1 - Éfficacité accrue pour l’amplification en chaîne par polymérase compétitive réelle mettant en oeuvre une pluralité de compétiteurs pour chaque cible - Google Patents

Éfficacité accrue pour l’amplification en chaîne par polymérase compétitive réelle mettant en oeuvre une pluralité de compétiteurs pour chaque cible Download PDF

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WO2011008818A1
WO2011008818A1 PCT/US2010/041919 US2010041919W WO2011008818A1 WO 2011008818 A1 WO2011008818 A1 WO 2011008818A1 US 2010041919 W US2010041919 W US 2010041919W WO 2011008818 A1 WO2011008818 A1 WO 2011008818A1
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pcr
competitor
nucleic acid
gene
competitors
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Aaron Barth Kantor
Timothy Stinchcombe
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IMMUNE TOLERANCE INSTITUTE Inc A CALIFORNIA NOT-FOR-PROFIT Corp
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IMMUNE TOLERANCE INSTITUTE Inc A CALIFORNIA NOT-FOR-PROFIT Corp
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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 present invention relates to real competitive PCR.
  • the present invention relates to methods of using multiple competitors per target in real competitive PCR.
  • Quantitative gene expression to screen for candidate biomarkers for disease progression or response to therapeutic intervention often begins with microarrays which permit the comparison of thousands of genes. Quantitative analysis of gene expression is often the second step in developing a validated, expressed gene marker for clinical monitoring or diagnostics.
  • single quantitative real-time PCR reactions e.g. Taqman
  • Single quantitative real-time PCR reactions have proven to be laborious and expensive, thereby limiting the number of genes that can be examined simultaneously.
  • Recent advances in the multiplexing of PCR reactions are circumventing this problem, allowing quantitative PCR to be used to monitor scores of genes at once.
  • the Sequenom mass spectrometry platform utilizing real competitive PCR, also known as quantitative, competitive end-point PCR is well suited for the application and has the potential to scale for very large numbers of samples.
  • Sequenom MassARRAY® platform as described in the product manuals as well as in Ding and Cantor (Proc.Natl.Acad.Sci.U.S.A 100 (6):3059- 3064, 2003), combines DNA amplification by quantitative end-point PCR with matrix- assisted-laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF- MS) to detect and quantitate specific DNA or RNA sequences. Determining the number of DNA or RNA molecules present in the starting reaction is one application of this technology.
  • DNA and a single synthetic competitor are amplified in a quantitative, competitive end-point PCR. This process is shown in FIGURE 1.
  • the initial PCR reaction consists of sample DNA to be measured (target) spiked with a synthetic DNA molecule (competitor) and primers that match both competitor and target.
  • the competitor matches the sequence of the target DNA region in all positions except a single nucleotide mismatch and serves as an internal standard.
  • PCR is performed and the PCR products from the competitor and DNA serve as templates for a second PCR primer extension reaction.
  • primer extension PCR e.g. Sequenom iPLEX
  • the products are measured by MALDI-TOF- MS. For this platform, single stranded nucleic acid molecules in the 1000 to 10,000 Dalton range (3-30 base pairs) need to be generated and deposited on a matrix.
  • the analyte-matrix mixture is irradiated by the laser to induce desorption and ionization.
  • the molecules pass through a flight tube to a detector. Separation occurs by time-of-flight and is proportional to the mass of the molecules.
  • the method measures the intrinsic property of the analytes at high speed (100 ⁇ s) and does not require extrinsic tags as needed for Taqman real-time PCR.
  • mass spectrometric analysis the peak areas of the distinct mass signals for the competitor and target extension products are calculated.
  • Non-linear regression analysis calculates target versus competitor concentration expressed as LOGEC50. EC50 represent the concentration at which the frequencies of target and competitor are equal.
  • a main advantage of the end-point competitive approach is optimization time required for each assay.
  • the range of target DNA or cDNA in a preparation can very substantially from sample to sample based on several factors including the specific target, quality of the sample preparation, level of expression, etc. Since the calculation of the number of DNA or cDNA molecules present in the starting reaction is the goal of the experiment, multipoint titration curves are usually run to ensure that the EC50 for all DNA targets in the reaction can be determined. The number of titration points and the difference in competitor concentrations between points will differ depending on the nature of the input DNA. For example, a 12-point titration with 1 :7 serial dilutions will cover the complete range transcript amounts per reaction (1 -282,000,000 molecules) while providing an accurate EC50.
  • serial dilutions with fewer serial dilutions can be employed successfully (i.e. 6 point titration with 1 :10 serial dilutions).
  • Serial dilutions of the competitor can be used to gain a broad dynamic range of at least nine logs.
  • Each titration point is represented by a single well on the PCR plate and a single well on the MALDI plate.
  • a 12-point titration of competitor requires 12 wells, each containing the sample DNA and the appropriate concentration of competitor DNA.
  • Vener, et al. (Biotechniques 21 (2):248-5, 1996) describes the use of four competitive standards at different concentrations with detection by gel electrophoresis. There is a large size difference among the gene and competitors ( ⁇ 100 bp). This paper only describes the detection of a single target gene.
  • the present invention provides for a method of performing real competitive PCR by co-amplifying multiple distinguishable masses with a single set of primers and extension, and detecting products of the multiple distinguishable masses.
  • the present invention further provides for a method of performing real competitive PCR by adding at least four distinct competitor DNAs in a single well containing at least one target nucleic acid, wherein all competitor DNAs have one stretch of nucleotides not homologous to the target nucleic acid, thereby reducing the number of wells required in the PCR assay, and performing real competitive PCR.
  • FIGURE 1 is a flow chart of the MassARRAY® quantitative gene assay amplification of competitor and target DNA
  • FIGURE 2 shows sequences of VIP genes and competitors
  • FIGURE 3 shows sequences of OAS genes and competitors
  • FIGURE 4 shows sequences of GADPH genes and competitors
  • FIGURE 5 shows sequences of MX1 genes and competitors
  • FIGURES 6A-6F show sequences of six genes with three competitors each;
  • FIGURE 7 shows mass spectra for the TBP gene with three competitors
  • FIGURE 8 shows the mass spectra of the six genes and competitors of
  • FIGURES 6A-6F are identical to FIGURES 6A-6F.
  • FIGURE 9 shows sequences from a sample assay.
  • the present invention provides a method of performing real competitive PCR using multiple, distinct, competitor DNAs in a single well containing at least one target nucleic acid to reduce the number of wells required in the assay.
  • the nucleic acid can be DNA or cDNA.
  • the method allows for detection of products of multiple distinguishable masses following co-amplification with a single set of primers and extension.
  • the method is performed with any suitable device that can perform quantitative end-point PCR for detection of products (also known as real competitive PCR) with matrix-assisted-laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF-MS) being a preferred method.
  • MALDI-TOF MS matrix-assisted-laser desorption/ionization, time-of-flight mass spectrometry
  • Detection by MALDI— TOF MS is a preferred method because PCR products can be the same length, which minimizes differences in PCR efficiencies between the target gene and the competitor standards.
  • One such device is the MassARRAY® described above; however, any other suitable platform can be used as determined by one skilled in the art. Any other method can be used that distinguishes products by mass, such as, but not limited to, separation by gel electrophoresis and detection by a variety of common methods (fluorescent, radioactive, chemiluminescent, etc.).
  • the competitor DNAs are each different from one another and from the target by one base pair.
  • the competitor DNAs are synthetic.
  • the total number of competitor DNAs in a single well can range between 1 and 12, and preferably 2 to 5 per well.
  • 4 PCR products, represented by 1 target and 3 competitors, are extended in the second amplification to result in a total of 4 different sequences, which molecules of different mass that can be measured by MALDI-TOF-MS.
  • the range of the competitor DNAs in a single PCR well (number of DNA molecules) can be optimized for each particular target and cover a broad or narrow range of concentrations.
  • the competitor DNAs are present in different concentrations in each well.
  • the assay can also include DNA quality control targets (i.e.
  • housekeeping genes which are independent of the target to aid in the evaluation of false negative and false positive results.
  • a key use of housekeeping genes is normalization across multiple samples. Housekeeping genes are always expressed because they code for proteins that are constantly required by the cell. They are essential to a cell and present under any conditions. It is also assumed that their expression is unaffected by experimental conditions, and therefore, they make good controls in an experiment.
  • the MALDI-TOF-MS method is capable of multiplexing at least up to 24 different targets each with a single competitor as described for the MassARRAY platform. That is, it has the capacity to distinguish at least up to 48 PCR products with different masses in the 1000 to 10,000 Dalton range. There is potential to analyze more targets. If more than one competitor is added per target per well, then the total number of targets per well assayed will have to be decreased since there is a finite capacity of MALDI-TOF-MS.
  • the table below provides an example of the trade-off in terms of multiplexing between number of targets and number of distinct competitors per target.
  • the present invention further provides for a method of performing real competitive PCR by adding at least four distinct competitor DNAs in a single well containing at least one target nucleic acid, wherein all competitor DNAs have one stretch of nucleotides not homologous to the target nucleic acid, thereby reducing the number of wells required in the PCR assay, and performing real competitive PCR.
  • This method is further described in Example 5, and is an example of using a higher number of competitor DNAs. This method is useful in obtaining more accurate results.
  • the method is performed as described above; however, this method also includes steps of binding two or more unextended primers to the competitor DNAs, extending by one mass modified ddNTP, and obtaining at least three additional unextended primer extension products.
  • the present invention also includes a kit for performing the method of real competitive PCR.
  • the kit preferably includes instruments for collecting a sample of nucleic acid (i.e. the target), such as swabs and syringes.
  • the kit can include shipping materials to ship the sample to a center for processing the assay.
  • the kit can include various competitor DNAs, primers, and solutions for performing the assay on site.
  • Blood tubes such as with the PAXGene® Blood RNA System (PreAnalytiX), can be included for RNA collection.
  • Newborn Screening can benefit from the multiplexed measurement of exogenous and endogenous DNA for detection of inherited (endogenous DNA) and infectious disease (exogenous DNA) using the multiple competitor method described above.
  • T cells one of the essential types of white blood cells that make up the immune system. While rearranging the receptor gene, the maturing T cell produces as a byproduct a DNA circle within the cell, called a T cell Receptor Excision Circle (TREC). These circles are stable, although they become diluted as T cells proliferate. Quantitative measurement of TRECs would enable diagnosis of all genetic forms of SCID. Quantitative real-time PCR of the delta-deletion TREC has been demonstrated.
  • the MALDI-TOF-MS modification described herein is particularly useful when the very high accuracy of the target concentration of TREC molecules is not needed.
  • TREC measurement by real-time PCR results in TREC copy number determinations that can be classified as positive or negative, even when TRECs are measurable, depending on the cutoff chosen from TREC copies.
  • Wisconsin School of Medicine and Public Health has presented real-time PCR data (APHL-2008) suggesting values up to 75 TREC copies/ ⁇ l of whole blood be considered negative for TRECs (positive for SCID).
  • APHL-2008 real-time PCR data
  • three or more different TREC competitors, at different concentrations in a single PCR well could represent 50 to 2000 TREC copies.
  • EC50 values can determine if the target is above or below 75 copies. Any sample giving a value below 75 would be classified as positive for SCID while those samples above 75 copies would be considered negative for SCID.
  • CMV infection infants with cytomegalovirus (CMV) infection are at risk for sensorineural hearing loss.
  • CMV infection is present in about 1 % of newborns of which 90% are asymptomatic. Early detection of CMV infection would enable closer monitoring and effective management for infants who develop hearing loss early in life.
  • CMV is a member of a large family of double stranded DNA viruses, herpes viruses, which have genomes of 100-200 genes and have been known to cause a variety of disease in humans.
  • Multiplexed, quantitative detection (MALDI-TOF-MS) of human herpes viruses, including CMV has been demonstrated. Additional multiplex screening for CMV, by inclusion of several different CMV specific competitors at different concentrations, in the same PCR well as used for TREC detection, adds value and cost-effectiveness to a newborn screen.
  • DNA from newborn blood can be collected with a variety of methods.
  • One preferred method is to use the same dried blood samples already collected from newborns (such Guthrie blood spot cards are currently collected in all U.S. states to test newborns for inherited diseases such as PKU and hypothyroidism).
  • DNAs isolated from Guthrie cards have previously been used for measurement of TRECs and CMV. Measurement of endogenous mRNA or exogenous viral mRNA, which are less stable than DNA, from such cards would be difficult and unlikely to be compatible with DNA measurement.
  • the quality of the DNA preparation is preferably assessed to aid in the evaluation of false negative and false positive results by inclusion of additional endogenous DNA targets ("housekeeping genes") that serve as protocol controls.
  • additional endogenous DNA targets include, but are not limited to, beta actin, beta-two microglobulin, and GAPDH (glyceraldehydes 3-phosphate dehydrogenase).
  • inclusion of an endogenous DNA target (or targets) in the multiplex can enable identification of non- informative assays due to poor DNA preparations.
  • Additional targets that can be added to newborn screening can include infectious diseases that are included in the TORCH panel and expanded TORCH panel when infections are suspected in newborns.
  • the TORCH panel consists of tests for antibodies to four organisms that cause congenital infections transmitted from mother to fetus. The name of the test is an acronym for the organisms detected by this panel: Toxoplasma gondii (toxoplasmosis), rubella (German measles), cytomegalovirus (CMV), and herpes simplex virus (HSV).
  • DNA viruses are particularly well suited for inclusion. Examples include, but are not limited to, herpes simplex virus (multiple), varicella-zoster virus (chicken pox), and Epstein Barr virus.
  • RNA viruses such as HIV and rubella (German measles) which have risk for the newborn can also be included.
  • EXAMPLE 3 Quantitative Gene Expression of Multiplex for MX1 , VIP, OAS, and GAPDH Housekeeping gene
  • the quantitative gene expression example assay below is for a multiple of 3 interferon-inducible genes plus a housekeeping normalization control.
  • Messenger RNA is transcribed to cDNA, which is then amplified with target specific primers. This assay is useful for monitoring patient response to IFN-beta therapy and the consequences of developing neutralizing antibodies to the drug (Pachner, et al. 2003 and 2006).
  • the best multiplex RT-PCR (TaqMan®) assays required two separate tubes, one for MXA (MX1 ) and GAPDH with FAM and VIC reporter dye systems and two for OAS and VIP also detected with FAM and VIC dyes (Pachner, et al. 2006). In general, running the normalization control (GAPDH) in a separate tube from the targets is not preferred.
  • a competitive endpoint PCR construction of the same four target gene expression assay, conducted in a single reaction well, is provided here in Table 3.
  • Assays are designed for multiplex measurements on the Sequenom MassARRAY platform by first inputting sequences into Sequenom proprietary software (QGE Assay Designer). The software determines appropriate PCR primer pairs, extension primer sequences (UEP or unextended primer sequence), and one competitor sequence per gene. The software also predicts the masses of the PCR extension products from the UEP binding to the gene of interest and the competitor sequence and extension of the UEP by one base complement to the gene or competitor. The predicted extension product masses are compared against actual mass measurements by the Sequenom mass spectrometer and the concentration of each gene in the original sample is determined (also by the Sequenom software).
  • QGE Assay Designer Sequenom proprietary software
  • the software determines appropriate PCR primer pairs, extension primer sequences (UEP or unextended primer sequence), and one competitor sequence per gene.
  • the software also predicts the masses of the PCR extension products from the UEP binding to the gene of interest and the competitor sequence and extension of the UEP by one base complement to the gene or competitor.
  • the predicted extension product masses are
  • FIGURES 2-5 Sequences used for QGE assays
  • Shown in FIGURES 2-5 are oligonucleotide sequences used for multiplex measurements of MX1 , VIP, OAS and GADPH house-keeping gene. The locations of PCR primers, competitor sequence and extension primers are shown. Also shown are the expected extension products with the predicted mass in Daltons.
  • the gene is shown on the top line, sense strand, in the conventional 5' to 3' direction and the 3' end may be continued at the end.
  • the PCR primers are underlined.
  • UEP sequence also referred to as unextended sequence primer or unextended primer, is shown under the target gene and each competitor. In some cases, the sequence shown is reverse complement (if the UEP unextended primer appears right of the competitor change then it is a reverse complement).
  • the final product that is detected in the mass spectrometer including its mass is shown on the next line.
  • a multiplex quantitative gene expression assay containing more than one competitor sequence for each gene represented in the multiplex.
  • a multiplex design is shown for MX1 , VIP, OAS and GAPDH (housekeeping gene).
  • the gene sequences were input to the Sequenom QGE Assay Design software and DNA sequences produced. Included in the output are PCR primers, a single competitor sequence with one base substitution from the native gene and one extension primer (UEP) used for a second PCR amplification.
  • the original Sequenom protocol calls for mixing native gene and single competitor sequence followed by PCR amplification of both sequences using the same primer set.
  • the amplified product is used as a template for primer extension in a second PCR reaction.
  • an extension primer binds to both native and competitor sequence just 3' or 5' to the base substitution designed by the software.
  • the extension primer is extended by one base pair that is complementary to the gene or competitor.
  • the Sequenom assay design software built the multiplex for MX1 , VIP, OAS and GAPDH.
  • the software designs one competitor sequence.
  • Two additional competitor sequences were added manually that varied by one base pair from the native sequence and the competitor sequence derived by Sequenom software.
  • the original UEP primer derived by the Sequenom software will also bind to the two additional competitors.
  • One skilled in the art can anticipate readily modifying the software to produce multiple competitors for the same target.
  • Sequenom software predicts the mass in Daltons of the unextended primer and the two extended primer products resulting from PCR amplification using the unextended primer and the native gene and Sequenom competitor. Additionally, with knowledge of the sequence of the two manually derived competitors and the Sequenom unextended primer one can predict the final sequence of the extended PCR products resulting from single strand PCR amplification using the unextended primer and the two manually derived competitors, in the same multiplex reaction. Since the masses are known for the mass modified dideoxynucleotides used for primer extension we can calculate the mass of the extension products from our manually derived competitors.
  • the mass modified dideoxynucleotides are (in Daltons): 271 for A, 247 for C, 287 for G and 327 for T.
  • Table 4 illustrates the mass differences are 16 Daltons between extended product that end in A versus G, 24 Daltons for A versus C, 40 Daltons for C versus G and 80 Daltons for T versus C and 56 Daltons for T versus A. Because the extended single base mass is constant the four extended products of the UEP for any one gene will always vary by the same amount.
  • the current illustration for the MX1 , VIP, OAS and GAPDH demonstrates that the mass difference between all products is sufficient for detection by MALDI- TOF mass spectrometry using the MassARRAY or other instruments (Table 5).
  • the mass separation between products should be at least 10, preferably 20 Daltons or more. Resolution is a function of the specific mass spectrometer (e.g., length flight path) and peak detection software.
  • Concentrations of the individual competitors would be determined by empirical titration. These should ideally span the range for the amount of each target.
  • the Sequenom QGE Assay Design software produces one competitor product and one UEP for each gene introduced into the multiplex. It does not support multiple competitors per gene for assay design. One has to manually design two additional competitors per gene for multiplexing with three competitors per gene. The masses of the extended products from the manually added competitors can be determined by simple math.
  • the Sequenom software allows manual input of multiple analyte sequences and masses which are not part of the original assay design by the software.
  • the analysis software will try to match the manual input and the analyte expected mass with actual masses measured. If the manually added analyte masses are matched to an actual mass measurement by MALDI-TOF then the mass peak is assigned a height and area by the software.
  • a multiplex real-competitive PCR was designed for six genes: GAPDH, TBP, UBC, IFN, IL-6, and TNF, using sequences obtained from public databases.
  • the competitor sequences for each gene are designed to vary by one base pair from the gene.
  • Extension primers are designed for each gene/competitor set using well known rules and/or public websites. The extension primer binds just 3' or 5' to the single base pair change between the gene and competitor.
  • FIGURES 6A-6F illustrate the sequences for six genes and with three competitors each for the unextended primer extension primer and the three extended primers with calculated masses are shown.
  • PCR primers are designated by green shading. Competitors are shown as reverse complement to the actual sequence input into the PCR reaction.
  • the real-competitive PCR involves addition of cDNA and competitors at different concentrations to a PCR cocktail in a single well. PCR amplification is performed using a PCR thermocycler. After PCR amplification of the genes and competitors, primers are added that bind just 3' or 5' to the one base pair difference between each gene and competitor. Another PCR reaction is performed in the presence of mass modified ddNTPs that terminate primer extension after one base pair addition to the primer.
  • FIGURE 7 shows the mass spectra for the TBP gene and with three competitors. The smallest mass difference of 16 Da difference between the 6002 Da and 5987 Da products is easily resolved
  • FIGURE 8 shows the mass spectra of all six genes and their competitors. Table 6 below lists the products by mass and the resulting intensity values. The peak area or peak height is reported by the mass spectrometer and each peak can be paired with the expected mass of each extension products. Since the molar concentration of each added competitor is known prior to the PCR and primer extension, the concentration of the gene of interest can be calculated.
  • the multiple competitor experiment can be setup such that the molar concentration of competitors does not span the molar concentration of the gene of interest. This is particularly important when cell stimulation results in thousands of fold increase in gene(s) copy number above non-stimulated conditions. If the gene of interest is not bracketed by the three multiple competitors, the EC50 is determined by less accurate extrapolation from the graph of the obtained data. Ideally the multiple competitor concentration should bracket the gene concentration. If the competitor concentrations do not bracket the gene concentrations then the experiment needs to be repeated with a different range of competitors that do bracket. More than three competitor concentrations per well could have advantages in reporting the EC50 accurately (more points for the graph) as well as ensuring that the gene of interest is bracketed in molar concentrations by the competitors.
  • This method expands on the method described above of three competitors with a single base mismatch to the gene of interest. It includes additional competitors (beyond three), at different molar concentrations. All competitors in the well have one stretch of nucleotides not homologous to the target gene. This mismatch is determined manually and requires that it is the same length of nucleotides as was found originally in the gene.
  • the mismatch can be any combination of nucleotides that have similar properties of the original nucleotides for GC content, melting temperature and meet standard PCR criteria for lack of primer/dimer formation, hairpins and long stretches of the same base (for instance 5 or more g nucleotides in a row), etc.
  • one stretch of mismatched nucleotides allows for a second unextended primer (termed UEP 2) to bind and extend by one mass modified ddNTP resulting in three additional UEP2 extension products, which differ by one base pair from each other.
  • the new UEP2 is designed to result in extended mass products that are unique to the multiplex.
  • FIGURE 9 An example assay is shown in FIGURE 9. Competitor sequences are shown as reverse complements of the actual competitor sequence. Key assay features include:
  • PCR primers 1 and 2 (TCCTATAAG GTTAGAAG G C and TTATTTCCTGGTTTAAT) bind and amplify all 6 competitors as well as the gene of interest.
  • UEP 1 (TAGTAGTTATGAGCCAGAG ) binds to the gene and competitors 1 ,2,3 and extends. UEP 1 cannot bind competitors 4,5,6 due to lack of homology.
  • UEP 2 (TGCTCACCCACCAACA ) binds to the gene and competitors 4,5,6 and extends. UEP 2 cannot bind competitor 1 ,2,3 due to lack of homology. [00086] 4.
  • the sequence ATCGATCGATCGATCGT contained in competitors 1 ,2, and 3 can be any sequence as long as it is not homologous to the gene or competitors 4,5,6 or the per primers
  • sequence catgcatgcatgcatgca contained in competitors 4-6 can be any sequence as long as it is not homologous to the gene or competitors 1 ,2,3 or the
  • CMV cytomegalovirus

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Abstract

La présente invention concerne un procédé pour réaliser l’amplification en chaîne par polymérase compétitive réelle par co-amplification d’une pluralité de masses distinguables avec un ensemble unique d’amorces et l’extension, puis détection des produits de la pluralité de masses distinguables. La présente invention concerne également un procédé pour réaliser l’amplification en chaîne par polymérase competitive par ajout d'au moins quatre ADN compétiteurs distincts dans un seul puits contenant au moins un acide nucléique cible, tous les ADN compétiteurs présentant un enchaînement de nucléotides non homologue de l’acide nucléique cible, réduisant ainsi le nombre de puits requis dans le dosage PCR. L'invention concerne également un procédé pour réaliser l'amplification en chaîne par polymérase compétitive.
PCT/US2010/041919 2009-07-14 2010-07-14 Éfficacité accrue pour l’amplification en chaîne par polymérase compétitive réelle mettant en oeuvre une pluralité de compétiteurs pour chaque cible Ceased WO2011008818A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042399A1 (fr) * 2020-08-31 2022-03-03 上海吉凯医学检验所有限公司 Procédé d'amplification génique et son application

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Publication number Priority date Publication date Assignee Title
US20030170684A1 (en) * 2000-02-07 2003-09-11 Jian-Bing Fan Multiplexed methylation detection methods
US7527930B2 (en) * 2005-01-21 2009-05-05 Gene Express, Inc. Compositions and methods of use of standardized mixtures for determining an amount of a nucleic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030170684A1 (en) * 2000-02-07 2003-09-11 Jian-Bing Fan Multiplexed methylation detection methods
US7527930B2 (en) * 2005-01-21 2009-05-05 Gene Express, Inc. Compositions and methods of use of standardized mixtures for determining an amount of a nucleic acid

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042399A1 (fr) * 2020-08-31 2022-03-03 上海吉凯医学检验所有限公司 Procédé d'amplification génique et son application

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