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WO1996009407A1 - Procede de quantification d'acides nucleiques - Google Patents

Procede de quantification d'acides nucleiques Download PDF

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Publication number
WO1996009407A1
WO1996009407A1 PCT/SE1995/001077 SE9501077W WO9609407A1 WO 1996009407 A1 WO1996009407 A1 WO 1996009407A1 SE 9501077 W SE9501077 W SE 9501077W WO 9609407 A1 WO9609407 A1 WO 9609407A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
process according
nucleic acids
competitor
target
Prior art date
Application number
PCT/SE1995/001077
Other languages
English (en)
Inventor
Peter Nilsson
Per-Åke Nygren
Mathias Uhlén
Björn PERSSON
Original Assignee
Pharmacia Biosensor Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE9403170A external-priority patent/SE9403170D0/xx
Priority claimed from SE9403169A external-priority patent/SE9403169D0/xx
Application filed by Pharmacia Biosensor Ab filed Critical Pharmacia Biosensor Ab
Publication of WO1996009407A1 publication Critical patent/WO1996009407A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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 nucleic acid analysis, and more particularly to a novel process for the quantification of nucleic acid fragments.
  • PCR polymerase chain reaction
  • PCR as well as of related nucleic acid amplification techniques, results in comparable amounts of product, starting either with few or many template copies. This limits the use of these techniques as quantitative tools. For many diseases, a quantitative measurement is needed to make proper diagnosis and it would be an advantage to be able to measure the amount of pathogen during treatment to make a relevant prognosis.
  • the use of competitive methods where titered amounts of an engineered and distinguishable competitor DNA are co-amplified with the wild type template enables a determination of the initial number of target templates.
  • the bottleneck of this strategy is the problems associated with specific detection of the respective PCR products for the determination of their relative ratios.
  • nucleic acid includes DNA and RNA.
  • competitive nucleic acid means a nucleic acid which is similar to the target nucleic acid and can be amplified using the same reagents, but which in some part differs from the target nucleic acid sequence to permit target and competitor nucleic acid fragments to be distinguished form each other by biospecific interactions.
  • the amplification step (ii) comprises using oligonucleotide primers capable of hybridising to sequences shared by the two nucleic acid species.
  • a typical application of the present invention comprises amplification of the sample nucleic acids in parallel with an amplification of a titered amount or a series of titered amounts of related nucleic acids, or competitor nucleic acids, recruiting the same reagents for amplification.
  • the amplification procedure can be any of several existing in vitro or in si tu nucleic acid amplification procedures (Abramson et al., (1993) Curr.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the resulting amplification product contains a mixture of amplicons originating from the target nucleic acids and the added competitor nucleic acid, and the numeral ratio of these resulting nucleic acid species reflects the initial stoichiometric relationship between the two nucleic acid species. Since the amount of competitor nucleic acids added is known, the initial number of target nucleic acids molecules can be determined if the ratio between the number of resulting amplicons can be determined. This determination can be performed employing hybridisations, fragment extensions or shortenings monitored by biospecific interaction analysis, preferably in real-time.
  • a basic feature of the present invention is that the amplified nucleic acid fragments are immobilized onto a suitable solid support utilizing a suitable coupling chemistry or biology.
  • suitable coupling chemistry or biology examples include the use of the high affinity interaction between biotin and streptavidin or avidin (Wilchek et al., Anal. Biochem. , 171: 1-32, (1988)), thiol coupling (Ljungqvist et al. , Eur. J. Biochem., 186: 557-561, (1989)) or interactions between an antibody and a hapten (Digoxigenin, Boehringer Mannheim, Germany) .
  • biosensor-based detection means to monitor, preferably in real-time, the further enzymatic or other manipulation of the immobilized nucleic acid fragments.
  • detection means may advantageously be taken from recent development in instrumentation for biospecific interaction analysis (BIA) , i.e. biosensor technology (J ⁇ nsson et al, Biotechniques, vol. 11, 5_: 620-627, (1991)) .
  • a biosensor may be defined as being a unique combination of a receptor for molecular recognition and a transducer for transmitting the interaction information to processable signals.
  • the receptor is supported on a sensing surface and molecular interaction with the receptor is detected as a change of a property of the sensing surface.
  • Exemplary of such biosensor technologies are those based on mass detecting methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods.
  • optical methods may particularly be mentioned those that detect mass surface concentration or refractive index, such as reflection-optical methods, including both internal and external reflection methods, e.g. ellipsometry and evanescent wave spectroscopy (EWS) , the latter including surface plasmon resonance spectroscopy (SPRS) , Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR) , evanescent wave ellipsometry, scattered total internal reflection (STIR), optical wave guide sensors, evanescent wave based imaging, such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, etc., as well as methods based on evanescent fluorescence (TIRF) and phosphorescence.
  • reflection-optical methods including both internal and external reflection methods, e.g. ellipsometry and evanescent wave spectroscopy (EWS) , the latter including surface plasmon resonance spectroscopy (SPRS) , Brewster angle refrac
  • BIAcore® J ⁇ nsson et al., (1991), supra
  • IAsysTM Hodgson et al., Bio/Technology, vol. 12, an. (1994)
  • SPR surface plasmon resonance
  • a fraction of, or the complete, sample e.g. amplification product
  • a suitable enzyme such as a restriction endonuclease or ribozyme.
  • the desired quantification of the ratios between the different nucleic acid species is obtained easily and reproducibly, since the activities of the enzymes can be monitored and controlled.
  • the quantification of nucleic acid fragments is performed by a first hybridisation.
  • the relative ratio between the nucleic acid species may be determined by quantitative monitoring of controlled primer extension using the immobilized nucleic acid fragments as templates, a suitable polymerase and free nucleotides.
  • Yet another embodiment of the present invention is based on the monitoring of sequence specific capture of DNA or RNA fragments via immobilized wildtype or competitor specific probes, followed by enzymatic extension or specific binding of another molecular species, such as antibody.
  • the invention will in the following be further illustrated by non-limiting examples with reference to the appended drawings.
  • FIG. 1 shows the sequences, lengths and relations of synthetic oligonucleotides used in some of the Examples.
  • the oligonucleotides are denoted 1 through 6.
  • the three subfragments can be assembled into a 69 bp double stranded DNA fragment via the 3 nt protrusion ends.
  • the G in oligonucleotide 4 marked with an arrow indicates the nucleotide determined in the mini DNA-sequencing protocol, in which a 5'-biotinylated version of oligonucleotide 6 was used.
  • Figure 2 is a schematic drawing of different strategies for PCR product quantification employing real ⁇ time monitoring of nucleic acid hybridisations.
  • the signal from the hybridisation reaction may be enhanced by binding of e.g. an antibody to a label present in the oligonucleotide used for hybridisation.
  • Figure 3 is a schematic view of the consecutive steps in the gene assembly mentioned in the description of Fig. 1.
  • Panel F shows a hybridisation of oligonucleotides enabling enzymatic extension. 5'-phosphate groups of the oligonucleotides are indicated (open ring) .
  • Figure 4 is a sensorgram from biosensor analysis of the gene assembly in Fig. 3. The capital letters correspond to the different events as outlined in Fig. 3.
  • FIG. 5 is a sensorgram from a DNA synthesis experiment.
  • the DNA polymerase activities of T7 DNA polymerase and E. coli DNA polymerase I (Klenow fragment) were investigated in real-time in a primer extension experiment.
  • Figure 6 shows a cleavage experiment with the endonuclease Xho I.
  • Figure 7 is a schematic drawing of the principles for enzymic quantification of PCR products obtained e.g from a competitive PCR strategy.
  • Figure 8 is a schematic drawing of the DNA strands which in Example 1 were present in different ratios as immobilized on the biosensor surface. Shown also are the nucleotide sequences of the two different oligonucleotides "comp” and "wt" (each is 17 nucleotides long) used for the hybridisation experiment.
  • Figure 9 is a histogram showing the relative responses obtained in the hybridisation experiment in Example 1, for the different flow cells of the biosensor.
  • Figure 10 is a sensorgram obtained in an experiment wherein the signal from a hybridisation (left) was amplified by the binding of an antibody (right) , recognizing label groups present in the 5'-ends of the oligonucleotides used for hybridisation in Example 4.
  • oligonucleotides designed for assembly into a 69 bp fragment (Fig. 1), were synthesized on an automated DNA synthesizer (Gene Assembler ® Plus, Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer's recommendations.
  • the oligonucleotides were purified using an FPLC® pepRPC5/5 column (Pharmacia Biotech AB) (Hultman et al, Biotechniques, 1: 84-93, (1991)). Phosphorylation was performed according to Sambrook et al (Molecular ⁇ cloning: A laboratory manual, Cold Spring Harbour Laboratory Press) .
  • Oligonucleotides 1 and 6 were synthesized both with and without a 5' biotin group using biotin-phosphoramidite (Clontech) , for use in different examples.
  • Other oligonucleotides used in the examples are listed below:
  • NIPE-8 Biotin-AACACAACGCTCTACAGCAGAATTGTGAGCGGATAACAATT-3 NIPE-18: JTC-CTCCTGCAGCTTCAAGAACTGTG-3 '
  • NIPE-17 Bl ⁇ tia-GTGATCTCCGTTTCCAATCCTGG-3' Wt: GGGAGAAAGAGTGTCTT-3 ' Comp: AATTGTTATCCGCTCAC-3
  • Running buffer HBS (10 mM Hepes, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA and 0.05% Tween® 20) or HBS ff ig ⁇ sa lt ( 0 mM Hepes (Sigma), pH 7.4, 0.5 M NaCl, 3.4 mM EDTA and 0.05% Tween® 20) .
  • HBS HBS with 0.15 M NaCl, 3.4 mM EDTA and 0.05% Tween® 20
  • HBS ff ig ⁇ sa lt 0 mM Hepes (Sigma), pH 7.4, 0.5 M NaCl, 3.4 mM EDTA and 0.05% Tween® 20
  • Ligation buffer One Phor-All-Buffer Plus, (Pharmacia Biotech AB) (10 mM Tris-acetate, pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate) supplemented with ATP and T4 DNA ligase (Pharmacia Biotech AB) to final concentrations of 1 mM and 0.1 Weiss U/ ⁇ l, respectively.
  • Extension buffer 0.05 U / ⁇ l polymerase in 28 mM Tris-HCl, pH 7.2, 30 mM citric acid, 10 mM MgCl2, 32 mM DTT, 4 mM MnCl2, supplemented with all four dNTPs to final concentrations of 0.2 mM.
  • Cleavage buffer 0.6 U/ ⁇ l of the endonuclease Xho I, in 10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM MgCl2 and 0.1 ⁇ g/ ⁇ l BSA (bovine serum albumin) . Antibody.
  • the antibody used in the hybridisation signal amplification experiment was mouse anti-fluorescein isothiocyanate (FITC) , DAK-FITC4 (DAKO, Denmark) .
  • FITC mouse anti-fluorescein isothiocyanate
  • DAK-FITC4 DAK-FITC4
  • the PCR-products to be quantified were obtained from PCR amplification of plasmid DNA harboring a wild type Chla ydiae tracho atis sequence or a corresponding sequence with an internal, genetically engineered, lac- operator sequence (competitor) (Fig. 8) .
  • the two resulting 97 bp long PCR products differ in an internal stretch of 21 nt, used as the specific hybridisation target sequences.
  • These PCR products were pre-mixed in four different ratios and subsequently immobilized onto a streptavidin Sensor Chip surface (SA5) , utilizing a biotin group present in one of the PCR primers.
  • SA5 streptavidin Sensor Chip surface
  • the immobilizations were performed by injections of 45 ⁇ l of the four different PCR product mixtures (200 nM DNA) in 0.5 M NaCl over the four surfaces at a flowrate of 1 ⁇ l/min. This resulted in the immobilization of approx. 2200 RU DNA in each flow cell (Fc) with calculated ratios between wild type DNA and competitor DNA of: Flow cell wt /competitor
  • the double stranded immobilized DNA was made single stranded using 1 mM HCl (10 ⁇ l at a flow rate of 5 ⁇ l/min) .
  • the efficiency of these strand separations were approximately 107%.
  • the high value can be explained by a simultaneous small loss of loosely bound double stranded DNA.
  • the streptavidin surface is almost unaffected, which is in contradiction to strand separation using NaOH (e.g. 50 mM) .
  • the subsequent hybridizations were performed at 25°C with an oligonucleotide concentration of 10 uM in HBS with 0.5 M NaCl (30 ⁇ l at a flowrate of 5 ⁇ l/min), using the wild type-specific oligonucleotide (wt) and the competitor- specific oligonucleotide (comp) , respectively, which were both 17 nucleotides long (Fig. 8) .
  • wt wild type-specific oligonucleotide
  • comp competitor- specific oligonucleotide
  • Each hybridisation was repeated seven times to allow a statistical evaluation of the results.
  • Regenerations of the single stranded templates were performed between each run using 1 mM HCl.
  • the hybridisations were performed at different oligonucleotide concentrations to assure a saturation of the single stranded templates.
  • the hybridisation strategy using surface plasmon resonance (SPR) detection shown here can be an alternative to methods based on electrophoresis or enzymatic detection principles. In order to enhance the signals obtained in the hybridisation, different methods can be used.
  • a template for hybridisation to the oligonucleotide NIPE-18 was obtained by a first immobilization (at a flowrate of 1 ⁇ l/min) onto a streptavidin coated sensor chip surface (SA-5) of approximately 2000 RU of a 97 PCR product (45 ⁇ l of 200 nM in 0.5 M NaCl) obtained from PCR on plasmid pRIT28-C.T., using oligonucleotides NIPE-17 and NIPE-18.
  • a single stranded template for the FITC-labeled oligonucleotide NIPE-18 was obtained.
  • a solution of NIPE-18 (2 ⁇ M) was subsequently injected, resulting in the hybridisation of 196 RU to the single stranded template.
  • a 300 nM solution of a mouse anti- FITC monoclonal antibody was injected (30 ⁇ l) .
  • the signal from a hybridisation can be amplified by at least a factor ten, by the use of a reagent capable of selectively recognizing a label present in the hybridising nucleic acid fragment.
  • oligonucleotides outlined in Fig. 1 can be assembled in a stepwise manner into a 69 bp double stranded DNA fragment from three smaller fragments with overlapping protrusions.
  • the experiments, outlined in Fig. 3 (A-E) were monitored in real-time using biosensor technology adapted for streptavidin-biotin chemistry. The corresponding sensorgram is shown in Fig. 4.
  • a prewashed sensor chip SA5 containing approximately 3500 RU of covalently coupled streptavidin was used to capture 5'- biotinylated oligonucleotide 1 (Fig. 3 (A)), injected (30 ⁇ l, 2 pmole/ ⁇ l) over the surface.
  • Fig. 3 (A) 5'- biotinylated oligonucleotide 1
  • oligonucleotides 4 (non-phosphorylated) and 3, were prehybridized and subsequently injected (40 ⁇ l, 2 pmole/ ⁇ l) over the surface.
  • This fragment contains a 3 nt protrusion which is complementary to the sequence present at the free end of the immobilized fragment, consisting of oligonucleotides 1 and 2.
  • No increase in the signal was observed when the injection was performed without DNA ligase (data not shown) .
  • T4 DNA ligase a significant binding was recorded by the real-time analysis, as demonstrated in Fig. 4 (C) .
  • the assembly was followed by strand specific elution of the non- biotinylated strand, (Fig. 3 (E) ) .
  • a short pulse of alkali (8 ⁇ l, 50 mM NaOH) resulted in a significant decrease in the signal (Fig. 4 (E) ) .
  • the amount released (1780 RU) suggests efficient release of the non-immobilized strand.
  • the amount of full length single stranded DNA present on the surface after this alkali pulse is approximately 970 RU. Note that the eluted DNA consists of two separate fragments, due to the absence of a phosphate group in oligonucleotide 4.
  • the resulting SPR response corresponds to a near complete extension of all the hybridized primers.
  • a final injection (8 ⁇ l) of alkali (50 mM NaCl) results in a decrease of approximately 680 RU, which correlates well with a complete release of the "second strand" DNA made up from extended primers (Fig. 5 (AB-III) ) .
  • DNA polymerase I (Klenow fragment) was analysed using the same experimental setup.
  • a gradual increase of the RU signal was seen only during the first minute of injection, after which the signal rapidly declines to a steady plateau value, (Fig. 5 (C-II) ) .
  • the subsequent SDS pulse only resulted in a minor decrease of the signal, (Fig. 5 (C) ) .
  • the resulting signal decrease after the final alkali injection corresponds to a complete release of the "second strand" DNA, obtained from an extension of the hybridized primers (approximately 680 RU) .
  • the complete dissociation from the immobilized templates seen for the Klenow polymerase already after approximately one minute of injection indicates that the DNA synthesis is completed after that period of time.
  • Oligonucleotide 1 contains the contribution from one strand to the recognition sequence for the endonuclease Xho I (Fig. 1) .
  • double stranded DNA obtained by extension towards the sensor chip surface using the priming outer oligonucleotide 6 together with Klenow polymerase, contains the complete recognition site (Fig. 6 (A) ) .
  • a 45 minutes pulse of Xho I present in its recommended assay buffer was injected over 1310 RU of such 69 bp substrate DNA (Fig. 6 (B) ) , obtained from a gene assembly experiment (data not shown) .

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Abstract

Procédé de quantification d'un acide nucléique cible dans un échantillon, qui consiste: (i) à ajouter à cet échantillon contenant ledit acid nucléique cible une quantité connue d'un acide nucléique concurrent; (ii) à amplifier les acides nucléiques cible et concurrent à l'aide de réactifs capables d'amplifier lesdits acides nucléiques concurrent et cible en parallèle; (iii) à immobiliser lesdits acides nucléiques amplifiés sur une surface de détection de biodétecteur; et (iv) à soumettre lesdits acides nucléiques immobilisés respectifs à une ou plusieurs interactions biospécifiques, puis à déterminer, à partir des modifications intervenues dans une propriété de la surface de détection provoquées par les interactions des acides nucléiques respectifs, les quantités relatives d'acides nucléiques cible et concurrent afin de déterminer ainsi la quantité dudit acide nucléique cible dans ledit échantillon.
PCT/SE1995/001077 1994-09-21 1995-09-21 Procede de quantification d'acides nucleiques WO1996009407A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE9403170-5 1994-09-21
SE9403170A SE9403170D0 (sv) 1994-09-21 1994-09-21 Process for nucleic acid hybridisation analysis
SE9403169A SE9403169D0 (sv) 1994-09-21 1994-09-21 Process for controlled manipulation of nucleic acid fragments
SE9403169-7 1994-09-21

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WO1996009407A1 true WO1996009407A1 (fr) 1996-03-28

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011253A3 (fr) * 1996-09-09 1998-05-07 Jose Remacle Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide
BE1010608A3 (fr) * 1996-09-09 1998-11-03 Jose Remacle Procede de quantification d'une sequence d'acides nucleiques.
WO1999014376A3 (fr) * 1997-09-19 1999-07-29 Genaco Biomedical Products Inc Detection de l'aneuploidie et deletion genique par co-amplification du dosage genique base sur l'acp de sequences a specificite chromosomique avec des agents synthetiques de regulation interne
WO2000046401A1 (fr) * 1999-02-03 2000-08-10 Lgc (Teddington) Limited Materiau de reference pour amplification d'acide nucleique
WO2001042496A3 (fr) * 1999-12-10 2001-12-27 Pyrosequencing Ab Methode de determination de la quantite d'acide nucleique dans un prelevement
WO2005007887A1 (fr) * 2003-07-15 2005-01-27 Daniel Henry Densham Mesure d'une reaction d'amplification de polynucleotide

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009156A1 (fr) * 1992-10-08 1994-04-28 The Regents Of The University Of California Dosages pcr permettant de determiner la presence et la concentration d'une cible

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009156A1 (fr) * 1992-10-08 1994-04-28 The Regents Of The University Of California Dosages pcr permettant de determiner la presence et la concentration d'une cible

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TIBTECH, Volume 9, October 1991, SCHWARZ et al., "Detection of Nucleic Acid Hybridization Using Surface Plasmon Resonance", pages 339-340. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011253A3 (fr) * 1996-09-09 1998-05-07 Jose Remacle Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide
BE1010608A3 (fr) * 1996-09-09 1998-11-03 Jose Remacle Procede de quantification d'une sequence d'acides nucleiques.
WO1999014376A3 (fr) * 1997-09-19 1999-07-29 Genaco Biomedical Products Inc Detection de l'aneuploidie et deletion genique par co-amplification du dosage genique base sur l'acp de sequences a specificite chromosomique avec des agents synthetiques de regulation interne
WO2000046401A1 (fr) * 1999-02-03 2000-08-10 Lgc (Teddington) Limited Materiau de reference pour amplification d'acide nucleique
WO2001042496A3 (fr) * 1999-12-10 2001-12-27 Pyrosequencing Ab Methode de determination de la quantite d'acide nucleique dans un prelevement
WO2005007887A1 (fr) * 2003-07-15 2005-01-27 Daniel Henry Densham Mesure d'une reaction d'amplification de polynucleotide
AU2004257918B2 (en) * 2003-07-15 2008-07-03 Daniel Henry Densham Measurement of a polynucleotide amplification reaction

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