WO1997021832A1 - Procede de determination de la presence de molecules d'acide nucleique en faible concentration - Google Patents

Procede de determination de la presence de molecules d'acide nucleique en faible concentration Download PDF

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Publication number
WO1997021832A1
WO1997021832A1 PCT/EP1996/005472 EP9605472W WO9721832A1 WO 1997021832 A1 WO1997021832 A1 WO 1997021832A1 EP 9605472 W EP9605472 W EP 9605472W WO 9721832 A1 WO9721832 A1 WO 9721832A1
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Prior art keywords
amplification
concentration
primers
primer
nucleic acid
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PCT/EP1996/005472
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German (de)
English (en)
Inventor
Manfred Eigen
Nils Walter
Petra Schwille
Frank OEHLENSCHLÄGER
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Evotec Biosystems Gmbh
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Publication date
Priority claimed from DE19545965A external-priority patent/DE19545965A1/de
Priority claimed from DE19634226A external-priority patent/DE19634226A1/de
Application filed by Evotec Biosystems Gmbh filed Critical Evotec Biosystems Gmbh
Publication of WO1997021832A1 publication Critical patent/WO1997021832A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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 a method for determining nucleic acid molecules in low concentration in samples to be examined by means of amplification reactions, in particular using polymerases and / or ligases, and unlabeled and detectable primers.
  • the 5'-nuclease assay (the so-called TaqMan polymerase chain reaction; Livak et al. In PCR Methods and Applications 4 (1995), 357-361) is similar to the method according to the invention.
  • the basis of this method is an oligonucleotide marked at the 5 ' end with a reporter molecule (fluorescein) and at the 3' end with a quencher molecule (rhodamine), which is also protected at the 3 'end by a phosphate residue against an enzymatic chain extension .
  • the probe In the course of the PCR reaction, the probe
  • Serious disadvantages of this method are the very complex and cost-intensive preparation of the double-labeled and end-group protected oligonucleotides, the involvement of enzymatic hydrolysis and the influence of media effects on the fluorescence yield, ie the quantifiability
  • the problem on which the invention is based is to provide a universally usable, easy-to-use and inexpensive method for the qualitative and quantitative detection of very small amounts of nucleic acid.
  • diagnosis of infectious pathogens it is desirable that the entire process can be carried out in compartments which are closed after the addition of the sample and do not have to be opened again in the course of the process
  • the method according to the invention is used for the determination of nuclear acid molecules in low concentrations in samples to be examined.
  • Amplification reactions are used. These use in particular polymerases and / or ligases. Unlabeled amplification primers are used. Detectable, in particular labeled primers, so-called detection primers, are also used. The detectable primers hybridize to the nucleic acid and / or are incorporated into the amplified nucleic acid by the amplification reaction, the concentration of the free detection primers decreasing. The decrease in this concentration and / or the increase in the concentration of the amplification products is measured and used as a measure of the presence of the nucleic acid molecule to be determined.
  • the detectable primers are preferably labeled with fluorescent groups.
  • primer-dependent reactions such as the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), isothermal 3SR (soap-sustained sequence replication), strand displacement amplification (SDA) and / or nucleic acid sequence-based amplification come as amplification reactions (NASBA) in consideration.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase PCR
  • SDA strand displacement amplification
  • LCR ligase chain reaction
  • the so-called gap LCR U. Landegren, Current Opinion in Biotechnology 1996, 7: 95-97
  • gap LCR U. Landegren, Current Opinion in Biotechnology 1996, 7: 95-97
  • fluorescence correlation spectroscopy FCS
  • FCS fluorescence correlation spectroscopy
  • Decrease in the concentration of the detection primer can no longer be determined or has reached a previously definable, expedient value.
  • differently labeled detection primers can be used.
  • the detection and evaluation can preferably be carried out using the FCS in the form of the cross correlation disclosed in WO 94/16313.
  • fluorescence-labeled primers it can be preferred in further embodiments of the method according to the invention to use a variant of fluorescence correlation spectroscopy using principles of near-field microscopy (WO 96/13744) or on analysis methods according to European patent application 96 116 373.0 to fall back on.
  • the latter application describes a method for analyzing samples by repeatedly measuring the number of photons per defined time interval of light which is emitted, scattered and / or reflected by the particles in the sample, and determining the distribution of the number of photons in the respective Time intervals.
  • the method is characterized in that the distribution of the specific brightness of the particles is determined from the distribution of the number of photons.
  • a polymerase without 5'-3'-exonuclease activity is preferably used. Otherwise, as in the Livak et al. reduced.
  • the polymerases are commercially available.
  • the detection primer it is also possible to dispense with an expansion of the detection primer during the method. This can be done, for example, by using a dideoxynucleotide at the 3 'end of the detection primer or by using labeled PNA as detection primers.
  • the increase in diffusion times is also in accordance with the invention of the detection primer by hybridization to the amplified nucleic acid sufficient for reproducible FCS detection.
  • the measure according to the invention to measure the decrease in the concentration of the detectable, in particular labeled, primers and to use it as a measure of the presence of the nucleic acid molecule to be determined, is based on the fact that at the beginning of the reaction the detection primer is in a state of high mobility while at the end of the reaction, the hybridization or the amplification reaction (incorporation into an amplification product) largely or completely converts it to a state of low mobility.
  • This change in mobility can be measured in a particularly suitable manner by the method of fluorescence correlation spectroscopy, it being possible for the sample to remain in the closed compartment.
  • the concentration of the amplification primer can be 5 to 200 times the concentration of the detection primer.
  • the number of fluorescent molecules, the relative ratio of fluorescence-labeled molecules of different sizes and their diffusion mobilities can be determined quickly and reliably from the correlation curve, which is preferably determined using fluorescence correlation spectroscopy.
  • the method according to the invention in the form of an end point titration and / or hybridization detects, by appropriate choice and concentration of the fluorescence-labeled primers, only the presence of the sought sequence, which has a sequence complementary to the detection primer.
  • the method is insensitive to unspecific amplifications.
  • a high concentration of amplification primer is desirable in order to ensure reliable amplification even of small amounts of nucleic acids to be detected.
  • the concentration of the amplification primer can be 0.5 ⁇ M
  • the detection primer is preferably present in a concentration of 1 to 10 nM.
  • sample carriers such as e.g. Polycarbonate films, which are characterized by the following advantages. they are
  • Foils with 96 or 384 compartments are preferred use.
  • the foils are filled with test liquid and sample liquid in approximately 5 to 20 ⁇ l portions and sealed.
  • the solutions are fixed as small drops by capillary forces in the upper part of the film, in which the compartment has a wall thickness of 10 to 20 ⁇ m required for the FCS method.
  • the foils can be fitted into a thermostatted frame. They are wetted with a thin layer of immersion liquid for the objective to be used in the FCS detection method.
  • the compartments can be used in particular for series of dilutions, screenings or also for different types of tests.
  • the typical measuring time for a film with 96 compartments is in the range of a few minutes up to a maximum of half an hour.
  • the quantitative determination of the nucleic acid to be detected can be carried out by using at least two amplification batches of the sample.
  • a dilution series of the first amplification batch is preferably produced.
  • the relative concentrations of the second and possibly further amplification batches result from the dilution ratio.
  • the detection concentration is known, ie the detection threshold of the detection system used, or another suitable threshold value, it is possible to quantify the nucleic acid to be detected by determining the reaction times or the number of amplification cycles until the threshold value is reached in the respective amplification approaches.
  • the threshold value should preferably be within the logarithmic amplification phase can be achieved in order to achieve a high level of quantification accuracy.
  • differences m in the amplification efficiency for example in the special structure of the nucleic acid to be detected, have an effect can be based, not on the measurement result
  • the method according to the invention is also suitable for the detection of point mutations, translocations, deletions or haplotype differentiation. It can also be used in the sense of a multiplex PCR.
  • inherited metabolic diseases such as, for example, the autosomal recessive cystic fibrosis
  • heterozygous patients can be distinguished from homozygous patients by using two detection primers with different dye markers.
  • detection of DNA and RNA genomes the latter e.g. by using an RT-PCR or 3SR reaction
  • FIG. 1 relates to a schematic representation of the method according to the invention using PCR.
  • FIG. 2 describes the shift in the correlation curve G (t) in the presence of the specified initial template quantities.
  • FIG. 4a shows the autocorrelation functions G (t) obtained in exemplary embodiment 1 by FCS analysis.
  • FIG. 4b shows the application of this data in a linearized form.
  • FIG. 5 illustrates the change in the relative diffusion times of the detection primer in the course of the amplification reaction according to exemplary embodiment 1.
  • FIGS. 6a and b illustrate the detection limit of the method according to the invention when applied to exemplary embodiment 1
  • FIG. 7 shows the use of a detection primer according to the invention during NASBA in exemplary embodiment 2.
  • FIG. 8 shows the location of the primers used within the gag region of the HIV-1 genome.
  • FIG. 9 shows a typical time course of the shift of the correlation curves due to the hybridization and extension of the detection primer in exemplary embodiment 2.
  • FIG. 10 illustrates the kinetics of the hybridization / extension reaction of the detection primer in exemplary embodiment 2.
  • FIG. 1 schematically describes the method according to the invention using PCR.
  • the amplification primers p1 and p2 used in the PCR are shown as arrows, the detection primer p3 is coupled to a fluorescent dye (circle).
  • the left part of the figure shows amplified artifacts in the absence of the template. This can e.g. are primer dimers of the highly concentrated primers pl and p2.
  • the right side of the figure shows p2 in the presence of the template. Only when the amplificates are present in a sufficiently high concentration does the amplificate act as a template for the low-concentration, fluorescence-labeled detection primer. The change in mobility of the primer can be clearly recognized by shifting the correlation curve.
  • FIGS. 2 and 3 Results obtained with the method according to the invention on Mycobacterium tuberculosis are shown in FIGS. 2 and 3.
  • the detection of this pathogenic pathogen previously took about 3 to 4 weeks due to the complex cell culture procedures. With the aid of the method according to the invention, this detection time can be greatly reduced.
  • FIG. 2 shows the shift in the correlation curve G (t) in the presence of the specified initial template quantities.
  • the number of template molecules specified in each case was initially presented at the start of the reaction with 50 .mu.l of reaction mixture, by the method according to the invention PCR cycles amplified and analyzed using the FCS method.
  • a template characteristic of the tuberculosis pathogen Mycobacterium tuberculosis was used.
  • the detection limit here was 100 template molecules per batch. The method is therefore sensitive enough to be used in any molecular diagnostic application.
  • FIG. 4a shows the auto-correlation functions G (t) obtained by FCS analysis with increasing number of PCR cycles of the target sequence IS6110 specific for Mycobacterium tuberculosis and M. bovis (exemplary embodiment 1).
  • the PCR amplification of a 106 bp segment starting from IO 5 strands of the genomic Mycobacterium tuberculosis DNA against a background of 2.5 ⁇ g unspecific human placenta DNA in 50 ⁇ l reaction volume, was carried out using the primer pair S1 / S2 as amplification primer and 10 nM TMR -labelled probe PRI as a detection primer (Table 1).
  • the detection primer binds between the amplification primers with the same orientation as the amplification primer.
  • FIG. 5 illustrates the change in the relative diffusion times of the detection primer in the course of the amplification reaction.
  • the diffusion times of the detection primer clearly reflect the PCR amplification chemistry with an initial lag phase before reaching the detection limit value, rapid exponential enrichment of the amplification products and a plateau phase. This plateau shows a saturation which is comparable to the end point of a titration.
  • the decreasing number of the molecules of the detection primer, which are in the laser focus on average, with increasing number of temperature cycles is presumably due to adsorption effects on the walls of the reaction vessels (secondary image in FIG. 5).
  • an apparent increase in emitted photons per molecule and second can be observed (secondary image in FIG. 5).
  • FIGS. 6a and 6b illustrate the detection limit of the method according to the invention when applied to exemplary embodiment 1.
  • the PCR amplification reactions were carried out using different amplification and detection primers (Table 1). Against a background of 2.5 ⁇ g unspecific human placenta DNA in a 50 ⁇ l reaction volume, a constant number of PCR cycles (either 36 or 40 cycles) were carried out, each with different starting concentrations on target genomes.
  • the detection limit is below 100 Mycobacterium tuberculosis genomes.
  • FIG. 7 shows the use of a detection primer according to the invention during the amplification reaction according to exemplary embodiment 2.
  • the method described by van Genem et al. (PCR Methods and applications 4, 177-184, 1995) NASBA method described was varied according to the invention as follows:
  • a detection primer (gagl) was added to the reaction mixture. This hybridized to a specific sequence of the 145 b RNA strand. Reverse transcription using AMV-RT produced a ds DNA molecule with 130 bp, while the amplification product produced using primers 1 and 2 had 167 bp.
  • the amplified RNA is detected and quantified using hybridization techniques such as ELGA (enzyme-imbed gel assay) and ECL (electrochemical luminescence). These techniques require the opening of the sample carrier with the risk of carryover contamination
  • FCS electrochemical luminescence
  • FIG. 8 shows the location of the primers used within the gag region of the HIV-1 genome.
  • FIG. 9 shows a typical time course of the shift in the correlation curves ( ⁇ 10 mm, 20 mm,
  • Example 1 the hybridized (gagl-145 b RNA) detection primer and the 130 bp components produced by extension were treated as one component due to their approximately equal diffusion times.
  • the correlation curves were filled using the equation known to the person skilled in the art to describe the autocorrelation function of a two-component system.
  • FIG. 10 illustrates the kinetics of the hybridization / extension reaction of the detection primer.
  • the meaning of the symbols used is as follows:
  • the value of the initial concentration of HIV-1 RNA molecules allows a distinction to be made between positive and false positive samples.
  • the direct quantitative analysis of the initial concentration of RNA molecules in a sample can be determined as follows.
  • the logarithm of the molecules contained in the sample represents a linear function of the incubation time necessary to achieve a certain degree of primer binding (secondary picture in FIG. 10).
  • the initial concentration of RNA molecules can thus be determined using a dilution series.
  • the combined approach of hybridization and extension of the detection primer was shown.
  • the data obtained using the FCS were confirmed by sequencing.
  • no non-specific binding of the detection primer could be determined, so that this makes it possible to dispense with an extension of the detection primer during the analyzes.
  • This can be done, for example, by using a dideoxynucleotide at the 3 'end of the detection primer or by using PNA primers.
  • the increase in the diffusion times of the detection primer by hybridization to the amplified RNA is also sufficient for reproducible FCS detection.
  • the polymerase chain reaction is used as the amplification method with a detection technique based on the fluorescence correlation spectroscopy (FCS) method using a detection primer labeled with fluorescent groups, e.g. an N, N, N ', N' -tetramethyl-5-carboxyrhodamm (TMR) -labeled detection primer.
  • FCS fluorescence correlation spectroscopy
  • the detection primers are incorporated into the amplified nucleic acid by the amplification reaction, the installation being accompanied by an increase in the diffusion time of the detection primer, which can be observed by means of FCS.
  • the oligodeoxynucleotides (Table 1) were purchased in HPLC-rem quality from NAPS (D-Göttingen).
  • the probes PRI, PR3, HS1 and HS3 were labeled with the 5-isomer of TMR at their 5 'end via an aminohexyl linker.
  • DOS [(10 x N / 86) x A 554 )] / [A 260 - (0.49 x A 554 )], where N is the number of bases m of the sample.
  • dNTPs 2'-Deoxynucleos ⁇ d-5'-triphosphates
  • Pharmacia the 5 ' -3 ' exonuclease-free fragment of the Thermus aquaticus DNA polymerase from Perkm-Elmer.
  • the human placenta DNA was purchased from Sigma. Genomic DNA from Mycobacterium tuberculosis was also used.
  • the amplification reaction was carried out either with 50 ⁇ l or 25 ⁇ l samples 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 2.5 mM MgCl 2 , 0.1 mg / ml gelatin, in each case 0 5 ⁇ M of the two amplification primers, in each case 200 ⁇ M of the four dNTPs, 50 ng / ⁇ l human placenta DNA as excess unspecific DNA, 1 to 20 nM of the TMR-labeled detection primer, and 0 05 U / ⁇ l of the 5'-3'-exonuclease-free fragment of the Thermus aquaticus DNA polymerase.
  • the amount of genomic Mycobacterium tuber cul osi s DNA (in the range from 0 to IO 6 molecules each 50 ⁇ l of the reaction mixture) is as indicated.
  • the PCR reaction took place in reaction tubes (multiple safecap tubes) from Sarstedt.
  • the following temperature program was used in a TRIO thermoblock cycler from Biometra (Gottingen): denaturation at 94 ° C for 30 s, polymer detection at 56 ° C (primer pair S1 / S2) or 60 ° C (primer pair B1B / B2B) for 20 s, and elongation at 72 ° C for 30 s, each for the specified number of cycles.
  • FCS measurement and determination of relative diffusion times Following the PCR, 10 ⁇ l of the reaction mixture were examined by means of FCS using a 63x1.2 water immersion objective. A detailed description of the FCS can be found in the article by Eigen and Rigler (Proc. Natl. Acad Sei USA 91.5740-5747). The autocorrelation function for a mixture of particles with fast and slow translation diffusion times is described by Thompson (Topics in Fluorescence Spectroscopy, Vol. I, Lakowicsz, JR (ed), Plenum 1991).
  • the isothermal amplification method NASBA nucleic acid sequence-based amplification
  • FCS detection method
  • the method according to the invention is used in this exemplary embodiment to detect a simulated HIV infection.
  • the TMR-labeled detection primer gagl was synthesized by NAPS (Göttingen).
  • the sample carriers with 96 reaction compartments were made in the precision engineering workshop of the Max Planck Institute for Biophysical Chemistry; each compartment has a volume of 40 ⁇ l and a wall thickness of 40 ⁇ m.
  • the plasma used was hepatitis B and C, CMV and HIV-1 negative.
  • nucleic acids Isolation of nucleic acids from the plasma: The nucleic acids were bound to activated silica (50 ⁇ l suspension, 1 mg / ml), which was added to the lysis mixture. After a washing and drying step, the nucleic acids were eluted with 50 ⁇ l elution buffer (1 mM Tris pH 8.5) and stored at -70 ° C.
  • NASBA amplification of the isolated HIV-1 RNA Typical reactions were carried out in a 20 ⁇ l reaction volume containing 40 mM Tris pH 8.5, 12 mM MgCl 2 , 42 mM KCI, 5 mM DTT, 15% v / v DMSO, in each case ImM dNTP, each 2mM NTP, 0.1 U RNase H, 0.1 ⁇ g / ⁇ l BSA, 40 U T7 RNA polymerase, 8 U AMV reverse transcriptase, 0.2 ⁇ M primer 1,2 and 5 ⁇ l isolated nucleic acid (containing 100, 200, 500, 2000 HIV-1 RNA molecules).
  • Primer 2 5'- AGT GGG GGG ACA TCA AGC AGC CAT GCA AA - 3 '(sense NT 1358-1386 from HXB2).
  • gagl 5'- TMR-GAG ACC ATC AAT GAG GAA GCT GCA GAA TGG GAT - 3 '(sense NT 1395-1427 from HXB2)
  • Oligodeoxynucleotide sequence (5 ' ⁇ 3') "Localis. T, m on (° C) c IS6110 h

Abstract

L'invention concerne un procédé de détermination de la présence de molécules d'acide nucléique en faible concentration, dans des échantillons à analyser, par des réactions d'amplification, à l'aide d'amorces non marquées utilisées comme amorces d'amplification et d'amorces détectables, notamment d'amorces marquées, utilisées comme amorces de détection, ainsi que notamment de polymérases et/ou de ligases. Les amorces détectables s'hybrident sur les produits d'amplification et la diminution de la concentration en amorces détectables, notamment marquées, et/ou l'augmentation de la concentration en produits d'amplification sont mesurées et les résultats obtenus servent de références pour déterminer la présence des molécules d'acide nucléique concernées.
PCT/EP1996/005472 1995-12-08 1996-12-06 Procede de determination de la presence de molecules d'acide nucleique en faible concentration WO1997021832A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19545965.2 1995-12-08
DE19545965A DE19545965A1 (de) 1995-12-08 1995-12-08 Verfahren zur Bestimmung von Nukleinsäuremolekülen in niedriger Konzentration
DE19634226A DE19634226A1 (de) 1996-08-24 1996-08-24 Verfahren zur Bestimmung von Nukleinsäuremolekülen in niedriger Konzentration
DE19634226.0 1996-08-24

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WO1997021832A1 true WO1997021832A1 (fr) 1997-06-19

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061720A2 (fr) * 1999-04-12 2000-10-19 Nanogen/Becton Dickinson Partnership Amorces d'amplification par deplacement de brins a allele specifique et procede associe
WO2001068680A2 (fr) * 2000-03-17 2001-09-20 Carl Zeiss Jena Gmbh Dispositif et procede d'analyse de principes actifs destines a agir sur les processus intracellulaires
EP1180549A1 (fr) * 2000-03-27 2002-02-20 Olympus Optical Co., Ltd. Procede de mesure de polymorphisme
US6365346B1 (en) 1998-02-18 2002-04-02 Dade Behring Inc. Quantitative determination of nucleic acid amplification products
US6778724B2 (en) 2000-11-28 2004-08-17 The Regents Of The University Of California Optical switching and sorting of biological samples and microparticles transported in a micro-fluidic device, including integrated bio-chip devices
US7745221B2 (en) 2003-08-28 2010-06-29 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US20130095489A1 (en) * 2010-05-04 2013-04-18 Centers For Disease Control And Prevention Process for detection of multidrug resistant tuberculosis using real-time pcr and high resolution melt analysis

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EP0731173A2 (fr) * 1995-03-10 1996-09-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Procédé pour la détection directe d'un nombre réduit de oligonucléotides

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6365346B1 (en) 1998-02-18 2002-04-02 Dade Behring Inc. Quantitative determination of nucleic acid amplification products
WO2000061720A2 (fr) * 1999-04-12 2000-10-19 Nanogen/Becton Dickinson Partnership Amorces d'amplification par deplacement de brins a allele specifique et procede associe
WO2000061720A3 (fr) * 1999-04-12 2001-01-18 Nanogen Becton Dickinson Partn Amorces d'amplification par deplacement de brins a allele specifique et procede associe
WO2001068680A2 (fr) * 2000-03-17 2001-09-20 Carl Zeiss Jena Gmbh Dispositif et procede d'analyse de principes actifs destines a agir sur les processus intracellulaires
WO2001068680A3 (fr) * 2000-03-17 2002-08-22 Zeiss Carl Jena Gmbh Dispositif et procede d'analyse de principes actifs destines a agir sur les processus intracellulaires
EP1180549A1 (fr) * 2000-03-27 2002-02-20 Olympus Optical Co., Ltd. Procede de mesure de polymorphisme
EP1180549A4 (fr) * 2000-03-27 2004-03-10 Olympus Optical Co Procede de mesure de polymorphisme
US6778724B2 (en) 2000-11-28 2004-08-17 The Regents Of The University Of California Optical switching and sorting of biological samples and microparticles transported in a micro-fluidic device, including integrated bio-chip devices
US7745221B2 (en) 2003-08-28 2010-06-29 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US8426209B2 (en) 2003-08-28 2013-04-23 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US20130095489A1 (en) * 2010-05-04 2013-04-18 Centers For Disease Control And Prevention Process for detection of multidrug resistant tuberculosis using real-time pcr and high resolution melt analysis

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