WO1998010097A2 - Procede et systeme permettant de detecter par fluorescence et en dichromie une molecule isolee et de determiner une masse et une concentration moleculaires - Google Patents

Procede et systeme permettant de detecter par fluorescence et en dichromie une molecule isolee et de determiner une masse et une concentration moleculaires Download PDF

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Publication number
WO1998010097A2
WO1998010097A2 PCT/US1997/013938 US9713938W WO9810097A2 WO 1998010097 A2 WO1998010097 A2 WO 1998010097A2 US 9713938 W US9713938 W US 9713938W WO 9810097 A2 WO9810097 A2 WO 9810097A2
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Prior art keywords
probes
fluorophore
sample
target
detection
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PCT/US1997/013938
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English (en)
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WO1998010097A3 (fr
Inventor
Alonso Castro
John Williams
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Pioneer Hi-Bred International, Inc.
The Regents Of The University Of California
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Application filed by Pioneer Hi-Bred International, Inc., The Regents Of The University Of California filed Critical Pioneer Hi-Bred International, Inc.
Priority to AU38291/97A priority Critical patent/AU3829197A/en
Publication of WO1998010097A2 publication Critical patent/WO1998010097A2/fr
Publication of WO1998010097A3 publication Critical patent/WO1998010097A3/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

Definitions

  • Figure 3 a and 3b are schematic diagrams of the sample container and capillary set up.
  • Figures 6a-d are schematic diagrams of how the detection of the presence of a conjugate in two zones can be translated into a histogram that will indicate the presence or absence of a target nucleic acid.
  • Figure 9 is a graph of the detection of four targets produced from a computer simulation as outlined in Example 9.
  • Figures lOa-c are graphs of the detection of 30 targets produced from a computer simulation as outlined in Example 11.
  • the number of copies of a gene in a cell can be detected with the present method by lysing the cells and treating the lysate with fluorophore-bearing probes as described herein.
  • an internal standard comprising an endogenous gene of known copy number is used as a standard.
  • the ratio of AB:CD will reveal the copy number of the transfected host cells. For example, if the known copy number of the internal standard is 2 copies per genome and the ratio of AB:CD is 5:1, then the copy number of the transfected gene is 10 copies per genome.
  • Probes to two separate fragments can be designed. If the fragments are joined by ligase or by genetic recombination in a cell, the detection of the two probes will occur simultaneously. Unjoined fragments will bind to one probe but not the other. Therefore, no simultaneous detection of the probes would be observed.
  • the present invention can be used to examine the genomic context of a transgene as well as detect genetic transpositions.
  • the detection of a genetic insertion, whether a transposon or a transgene can be determined by hybridization of oligomer probes to the genomic target sequence subsequent to the incubation of the test sample with restriction endonucleases.
  • the presence of a transgene or a transposon can be detected by the increase in molecular weight of the target restriction fragment in the test sample. If there is no change in the mass of the genomic target fragment, the transgene or transposon has not inserted into this restriction fragment. If there is a decrease in mass of the fragment, the transposon has been removed from the site.
  • the insertion or deletion of a transgene or transposon can be detected if one probe hybridizes to the genomic target sequence and the other probe hybridizes to the transposon.
  • a coherent signal is obtained only when the transposon is inserted in the target sequence.
  • Single-nucleotide polymorphism can be detected according to whether at least one of the two target probes has a perfect match or is mismatched (Dulay et al., Poster #P-1110-T presented at the HPLC 96 meeting in San Francisco, CA on June 18, 1996). Multiplex detection of individual gene targets can be accomplished by the present invention.
  • multiple oligomer probes to several different target nucleic acids permit the detection of multiple gene targets in the same test sample.
  • Multiplex detection using degenerate or partially degenerate probes could be used to determine the level of genetic identity in genetically uncharacterized organisms.
  • Degenerate probes can be designed to hybridize to the 3' and 5' ends of repeated sequences so as to detect an undetermined number of the repeated sequence, many copies of which are dispersed through out the genome (e.g., Alu sequences in the human genome). A complex "fingerprint" would result from the analysis.
  • the target molecules can be nucleic acids, nucleic acid fragments, and other nucleic acid-like species that have specific nucleobase sequences (herein referred to as
  • a single target sequence can be monitored and femtomolar or lower concentrations of the target sequence within a test sample may be detected. If necessary, samples containing a high concentration of target nucleic acid can be diluted to a suitable concentration before hybridization to oligomer probes.
  • the nucleic acids used in the invention may be comprised of DNA or RNA (or both) having any of the naturally occurring bases (A, T, G, C, and U), non-naturally occurring bases (e.g., I), and modifications thereof.
  • the target nucleic acid can be modified in the sugar unit or internucleoside
  • the internucleoside linkages can be modified in any of a variety of ways, including, but not limited to, phosphorothioate, phosphorodithioate, alkyl- and aryl-phosphonate and phosphonothioate, carbamate, etc.
  • the target and probes be sufficiently long and of such chemical make-up that the two fluorophore-bearing probes can specifically hybridize to the target under conditions suitable for electrokinetic or mechanically induced flow of the test sample through the capillary.
  • the target nucleic acid is a naturally occurring DNA (e.g., a gene or fragment thereof) or RNA (e.g., mRNA) polymer.
  • the oligomer probes hybridize to target nucleic acids to form duplexes, triplexes or higher order structures. In a most preferred embodiment the oligomer probes hybridize to denatured RNA or DNA molecules to form a duplex.
  • Each oligomer probe has a fluorophore attached to it, preferably at the oligomer' s 3' or 5' end.
  • fluorophore preferably any relatively small fluorophore may be used.
  • Preferred fluorophores are fluoroscein, dansyl, fluoroscamine, OP A, NDA, rhodamine 6G, JOE, FAM, Texas Red, Cy5, Cy7, IRD41 and Bodipy-TR.
  • Oligomer probes according to the present invention preferably have between 8 to 50 nucleobases, with 10 to 25 nucleobases preferred. Obviously, the length of the oligomer probe chosen will depend on the target nucleic acid sought as well as the conditions chosen to conduct hybridization of probe to target. It is a routine matter for one of skill in the art to choose the appropriate length oligomer probe suitable for the desired purpose.
  • the determination of the molecular weight of the target nucleic acid requires the use of one or more external standards that have different molecular weights.
  • a plurality of external standards with molecular weights are provided.
  • capillary electrophoresis e.g., ⁇ Hindlll fragments, 123
  • bp DNA ladder or 1 kb ladder, Life Technologies, Inc, Gaithersburg, MD
  • bp DNA ladder or 1 kb ladder, Life Technologies, Inc, Gaithersburg, MD
  • each vertical line (or peak) in Figure 4 represents the signal (i.e., a burst of photons) from one or more fluorophores (A or B) in one of the zones as it passes through the zone at a particular time.
  • the simultaneous presence of a peak in line A and line B indicates the presence of the target oligonucleotide to which both oligomer probes are bound.
  • Data interpretation may be done by any convenient method. In one
  • time interval analysis one measures the time ( ⁇ t jk ) between each
  • ⁇ t max is an arbitrary time chosen to be greater than the suspected
  • ⁇ t jk in the particular migration time range (e.g., 0-5 msec, 5-10 msec, 10-15 msec,
  • Figure 6 A shows the
  • Detectors 1 and 1' detect the presence of a first fluorophore in zones j and k,
  • detectors 2 and 2' detect the presence of a second fluorophore in
  • the horizontal direction is a time axis.
  • ⁇ t conj conjugate, ⁇ t conj .
  • ⁇ t conj is usually not known, but ⁇ t max can be chosen
  • ⁇ t C011J « 2.2 ms and ⁇ t max 7 ms.
  • ⁇ t jk are
  • Fig. 6B Each row is a simultaneous emission in zone j, and each column is a simultaneous emission in zone k. Each cell represents the time between those simultaneous emissions in each row and column in which the cell resides.
  • a histogram is created.
  • a "bin" time width is chosen. In Fig. 6C the bin time width is 1 ms and in Fig. 6D it is 0.5 ms. Then the
  • ⁇ t Jk number of ⁇ t Jk in a particular bin (e.g., 0-1 ms, 1-2 ms, 2-3 ms, etc., for Fig. 6C, and 0-
  • Other embodiments include a greater number of detection sites such that the target oligonucleotide can be detected at three or more spatially separated detection zones.
  • the output from multiple detectors can be correlated as previously discussed using migration velocities between zones 1 and 2, 2 and 3, etc. Improved signal-to- noise ratio can be achieved in the same amount of time as when two detection zones are used because there will be 2, 3 or more data points (_t l ⁇ 2 ,_t 2 ⁇ 3 etc.) obtained.
  • sample volume required to fill the sample compartment is ⁇ 3 ml, but accommodation of smaller sample volumes can be accomplished by reducing the scale of the sample
  • the capillary is square, but other geometric configurations, such as rectangular or cylindrical, can be used also.
  • a high- voltage power supply is connected to the sample reservoirs by means of platinum electrodes, for example. After the sample containing the target nucleic acid is placed in the sample reservoir a voltage is applied to induce electrophoretic migration through the detection zones. In a preferred embodiment 200 V/cm are applied, but other voltages in the range 10-1000 V/cm are appropriate.
  • Figure 2 shows a general schematic diagram for the detection apparatus of the present invention. Multiple lasers can, of course, be used, but preferably only a single laser is used as the excitation source. In some cases, all fluorophores can be excited simultaneously by the same laser light source.
  • Certain fluorophores do not have a large enough Stokes shift, however, making it necessary to use multiple light sources emitting different wavelengths to excite different fluorophores.
  • the laser is attenuated by a variable neutral density filter and then split into two parallel beams by a beam splitter-mirror combination.
  • the resulting 1-5 mW laser beams are focused by a focusing lens into the sample capillary cell by an achromatic lens to yield two detection zones (defined by the region within the capillary in which the focused laser beam is of sufficient intensity to excite the fluorophores) separated by a given distance.
  • the zones are about 10 ⁇ m in diameter and
  • the laser is pulsed and the detectors gated off during each pulse. This eliminates detection of Rayleigh and Raman scattering, which occur within the duration of the laser pulse.
  • the detectors are gated on between the pulses to detect fluorescence emission, which generally has a longer half-life than Rayleigh and Raman scattering.
  • the laser pulse rate should be set by considering, inter alia, the intensity of the laser, the fluorescence half-life of the fluorophores, and photobleaching effects. A suitable and typical pulse rate is about 80 MHz. Those skilled in the art will appreciate that selection of the pulse pattern and the laser wavelength depend on the particular fluorophores being used.
  • fluorophores with large Stokes shifts or which emit at wavelengths in the infrared region of the electromagnetic spectrum may obviate the need for time gated detection.
  • IRD41 Li-Cor, Lincoln, NE
  • the laser wavelength must be selected that will excite the fluorophore to fluoresce at a level that will be detectable by the optics and electronics of the system.
  • the fluorophores of the probes absorb the incident light, which causes them to enter an excited electronic state. As they relax from this excited state to the ground electronic state the fluorophores emit a characteristic wavelength. There will be two separate emission intensities and wavelengths corresponding to the two separate fluorophores hybridized to the target nucleic acid.
  • the length of the zone (i.e., the dimension in the direction of flow) should be adjusted to maximize sensitivity of the method.
  • the longer the zone the more excitation and emission cycles each fluorophore undergoes. But by increasing the zone length, one also increases the likelihood that two unbound fluorophores will simultaneously pass through the zone and emit, leading to false positive signals. False positive signals will also arise when the sample is too concentrated, since the more concentrated the solution the greater the likelihood that two unbound fluorophores will simultaneously pass through the detection zones. It is a routine matter for the skilled artisan to adjust the laser intensity, the size of the detection zone and the concentration (e.g., by diluting it) to obtain a suitable (if not maximal) signal-to-noise ratio.
  • Each fluorescent emission is then spectrally filtered by a bandpass interference filter, optimized to discriminate among fluorophores, and detected by photodiodes.
  • Suitable parameters and apparatuses include a 40x, 0.75 NA microscope objective, a 0.4 x 0.4 mm square slit that defines a 10 x 10 ⁇ m detection area for each laser beam,
  • photodetector that is a E.G.& G single photon avalanche photodiode. It is also possible to image both detection zones with one objective lens and one set of detectors. It will be recognized that alternate microscope objectives, slit geometries and widths, and detectors can be used and are within the spirit of the present invention. It will be appreciated that the particular optics are not critical features of the invention and any combination of optical elements, including optical fibers that allow simultaneous detection of one or more detection zones, can be used.
  • Each detector output signal is analyzed by independent time correlated single- photon-counting electronics under computer control.
  • the time correlated single photon counting technique allows the determination of the arrival time of single photons to the detector, measured with respect to the arrival time of the laser pulse to the sample (See e.g. O'Connor and Phillips, Time Correlated Single Photon Counting, Academic Press (1984)).
  • the detection electronics reject Raman and Rayleigh scattering by using a time-gated window set such that only delayed fluorescence photons are detected, thus increasing the signal-to-noise ratio of single-molecule detection.
  • fluorescence data are collected at 1 ms intervals.
  • the ability to distinguish photon bursts from each fluorophore depends not only on the concentration but the bin size, which is the time period during which photons emitted by the fluorophore are counted. It is a routine matter for the artisan to adjust these parameters to yield a suitable (if not maximal) signal-to-noise ratio.
  • Typical bin sizes are 0.10 to 0.05 of the fluorescent burst width.
  • the fluorescence burst width is the collection of photons emitted by a single molecule as it traverses a laser beam.
  • the burst shape is a convolution between the molecular velocity function (usually linear) and the laser intensity profile (usually a gaussian function). The width is measured at half the amplitude of the burst (i.e. FHWM).
  • the burst FWHM is typically about 10 to 20 msec.
  • chloroform:isoamyl alcohol 24:1 was added and the mixture shaken for five minutes.
  • the tube was centrifuged for 20 minutes at 1100 x g and about 15 ml of supernatant recovered by aspiration with a pipet.
  • the contents of the tube were mixed by inversion, the tube was let stand at room temperature for 30 minutes and centrifuged for 10 minutes at 1100 x g.
  • the DNA was collected with a glass stirring rod
  • the DNA was dissolved in 200 ⁇ l of a 0.1 mM NaEDTA
  • concentration of the DNA was approximately 5 ⁇ g/ ⁇ l.
  • PNA probes were purchased from PerSeptive Biosystems, Inc.
  • SEQ ID NO.2 5'-GCC AGT GTT GTA CCA-Lys-BodipyTR-3*
  • Lys is the amino acid lysine; Rho (rhodamine 6G), and BodipyTR (Bodipy Texas
  • a Probe Mix of Seq ID No. 1 and Seq. ID No. 2 was made by diluting l ⁇ l of
  • each probe (which had been dissolved in lOmM Tris-Cl and 0.1 mM NaEDTA to a concentration of 50 ⁇ M) in 2.5 mL water so that the final concentration of each probe
  • the Probe Mix was 20 nM.
  • the Probe Mix was vortexed and placed in -20°C freezer until use.
  • Figure 5 demonstrates the detection of the BT toxin gene in a single detection zone.
  • SEQ ID NO. 3 5'-TAT TTG ACG TGG TTT-Lys-Rho-3'
  • SEQ ID NO. 4 5'-BodipyTR-0-GCC-TCC-ACG-CAC-GTT-
  • Figure 15 demonstrates the detection of the ⁇ target in a single detection zone.
  • the Monte Carlo algorithm consisted of 104857600 steps, each 5 x 10 "6 sec in duration. During each step, the following events may or may not have occurred, consistent with their respective probabilities.
  • the signal-to-noise ratio must be greater than 3.
  • a signal-to-noise ratio of 30 was obtained. This means that even when the velocity of one target is as high as 8 times that of another, the slower moving target can easily be detected, although with a reduced signal-to-noise ratio (i.e., 100 for the first experiment versus 30 in this case).
  • the model sample contained the thirty targets listed in the table below:
  • the detection zone volume is described by the shape of the focused laser beam in the capillary, in terms of its height along the capillary axis and the cross-sectional area perpendicular to this axis.
  • counting efficiency was always held constant as the detection geometry was varied. As the volume was increased the sample concentration was reduced according to Poisson probability in order to avoid a loss in efficiency due to the presence of multiple targets being present at the detector at the same time.
  • the analysis time increased linearly with the detection zone height.
  • the constraint on counting efficiency forced a reduction in sample concentration as the detection zone volume increased.
  • the signal-to-noise ratio was constant at about 19 for all data points.
  • the cross-sectional area was varied between 400 and

Abstract

La présente invention concerne un nouveau procédé qui permet de détecter la présence d'une molécule cible dans un échantillon et d'en déterminer la masse et la concentration moléculaires. Le procédé consiste à déterminer les vitesses électrocinétiques en mesurant le temps nécessaire pour que des molécules isolées, marquées au moins avec deux sondes fluorescentes, parcourent une distance donnée entre deux faisceaux laser. Une comparaison entre la vitesse d'électrophorèse qu'on sait caractéristique d'étalons particuliers de masse moléculaire et la vitesse de l'espèce-cible permet de déterminer la masse moléculaire de la cible. Dans une autre forme d'exécution, on peut identifier une molécule cible dans un échantillon pour essai complexe par un procédé double palpeur de zone de détection d'une molécule isolée mettant en oeuvre un écoulement mécanique, électroosmotique ou électrocinétique d'un échantillon à travers un tube de transport. Le procédé est extrêmement sensible et permet de détecter et de déterminer en quelques secondes la masse moléculaire d'une molécule cible présente dans une concentration aussi petite qu'une femtomole.
PCT/US1997/013938 1996-09-03 1997-08-08 Procede et systeme permettant de detecter par fluorescence et en dichromie une molecule isolee et de determiner une masse et une concentration moleculaires WO1998010097A2 (fr)

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AU38291/97A AU3829197A (en) 1996-09-03 1997-08-08 Method and apparatus for single molecule two color fluorescent detection and molecular weight and concentration determination

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US08/709,468 1996-09-03

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Cited By (16)

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US6263286B1 (en) 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
WO2002099398A1 (fr) * 2001-06-06 2002-12-12 U.S. Genomics, Inc. Procedes et appareil pour la caracterisation de polymeres individuels
US6762059B2 (en) * 1999-08-13 2004-07-13 U.S. Genomics, Inc. Methods and apparatuses for characterization of single polymers
EP1436426A2 (fr) * 2001-10-24 2004-07-14 Singulex, Inc. Procedes destines a detecter des haplotypes genetiques par interaction avec des sondes
WO2004066185A1 (fr) * 2003-01-23 2004-08-05 U.S. Genomics, Inc. Methodes d'analyse de populations de polymeres
US7262859B2 (en) 2004-10-13 2007-08-28 U.S. Genomics, Inc. Systems and methods for measurement optimization
US7282330B2 (en) 2002-05-28 2007-10-16 U.S. Genomics, Inc. Methods and apparati using single polymer analysis
US7351538B2 (en) 2004-08-23 2008-04-01 U.S. Genomics Systems and methods for detecting and analyzing polymers
US7371520B2 (en) 2002-05-28 2008-05-13 U.S. Genomics, Inc. Methods and apparati using single polymer analysis
US7595160B2 (en) 2004-01-13 2009-09-29 U.S. Genomics, Inc. Analyte detection using barcoded polymers
US7977048B2 (en) 2004-01-13 2011-07-12 Pathogenetix, Inc. Detection and quantification of analytes in solution using polymers
US8045140B2 (en) * 2007-10-18 2011-10-25 Stc.Unm Method for multivariate analysis of confocal temporal image sequences for velocity estimation
US8168380B2 (en) 1997-02-12 2012-05-01 Life Technologies Corporation Methods and products for analyzing polymers
US8518705B2 (en) 1999-08-13 2013-08-27 Pathogenetix, Inc. Methods and apparatuses for stretching polymers
US9028776B2 (en) 2012-04-18 2015-05-12 Toxic Report Llc Device for stretching a polymer in a fluid sample
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis

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Cited By (22)

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US8168380B2 (en) 1997-02-12 2012-05-01 Life Technologies Corporation Methods and products for analyzing polymers
US6772070B2 (en) 1998-08-13 2004-08-03 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6263286B1 (en) 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6927065B2 (en) 1999-08-13 2005-08-09 U.S. Genomics, Inc. Methods and apparatus for characterization of single polymers
US8518705B2 (en) 1999-08-13 2013-08-27 Pathogenetix, Inc. Methods and apparatuses for stretching polymers
US6762059B2 (en) * 1999-08-13 2004-07-13 U.S. Genomics, Inc. Methods and apparatuses for characterization of single polymers
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
EP1402245A4 (fr) * 2001-06-06 2006-03-22 Us Genomics Inc Procedes et appareil pour la caracterisation de polymeres individuels
EP1402245A1 (fr) * 2001-06-06 2004-03-31 U.S. Genomics, Inc. Procedes et appareil pour la caracterisation de polymeres individuels
WO2002099398A1 (fr) * 2001-06-06 2002-12-12 U.S. Genomics, Inc. Procedes et appareil pour la caracterisation de polymeres individuels
EP1436426A4 (fr) * 2001-10-24 2005-11-02 Singulex Inc Procedes destines a detecter des haplotypes genetiques par interaction avec des sondes
EP1436426A2 (fr) * 2001-10-24 2004-07-14 Singulex, Inc. Procedes destines a detecter des haplotypes genetiques par interaction avec des sondes
US7282330B2 (en) 2002-05-28 2007-10-16 U.S. Genomics, Inc. Methods and apparati using single polymer analysis
US7371520B2 (en) 2002-05-28 2008-05-13 U.S. Genomics, Inc. Methods and apparati using single polymer analysis
WO2004066185A1 (fr) * 2003-01-23 2004-08-05 U.S. Genomics, Inc. Methodes d'analyse de populations de polymeres
US7595160B2 (en) 2004-01-13 2009-09-29 U.S. Genomics, Inc. Analyte detection using barcoded polymers
US7977048B2 (en) 2004-01-13 2011-07-12 Pathogenetix, Inc. Detection and quantification of analytes in solution using polymers
US7402422B2 (en) 2004-08-23 2008-07-22 U.S. Genomics, Inc. Systems and methods for detecting and analyzing polymers
US7351538B2 (en) 2004-08-23 2008-04-01 U.S. Genomics Systems and methods for detecting and analyzing polymers
US7262859B2 (en) 2004-10-13 2007-08-28 U.S. Genomics, Inc. Systems and methods for measurement optimization
US8045140B2 (en) * 2007-10-18 2011-10-25 Stc.Unm Method for multivariate analysis of confocal temporal image sequences for velocity estimation
US9028776B2 (en) 2012-04-18 2015-05-12 Toxic Report Llc Device for stretching a polymer in a fluid sample

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CR5602A (es) 1998-08-31
WO1998010097A3 (fr) 2002-10-17
AR009477A1 (es) 2000-04-26

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