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WO2001090407A1 - Mesure quantitative de quantites de molecules dans des melanges complexes - Google Patents

Mesure quantitative de quantites de molecules dans des melanges complexes Download PDF

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Publication number
WO2001090407A1
WO2001090407A1 PCT/EP2001/006009 EP0106009W WO0190407A1 WO 2001090407 A1 WO2001090407 A1 WO 2001090407A1 EP 0106009 W EP0106009 W EP 0106009W WO 0190407 A1 WO0190407 A1 WO 0190407A1
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WO
WIPO (PCT)
Prior art keywords
molecules
sample
measurement
molecular
complex
Prior art date
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Ceased
Application number
PCT/EP2001/006009
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German (de)
English (en)
Inventor
Johannes Schuchhardt
Dieter Beule
Holger Eickhoff
Hanspeter Herzel
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MICRODISCOVERY GmbH
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MICRODISCOVERY GmbH
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
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Publication of WO2001090407A1 publication Critical patent/WO2001090407A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to a method for the quantitative measurement of molecular amounts in complex mixtures by means of microarrays and other microstructured carriers. Fields of application of the invention are molecular biology, medicine and the pharmaceutical industry.
  • Microarrays consist of a large number of polymeric molecules that are firmly attached to the surface of a carrier substrate with a defined geometric arrangement. Microarrays of polynucleotides and polypeptides have become an important tool in molecular biology as well as related technologies and industries. They are used in a number of applications, for example in screening, in DNA sequencing and in gene expression analysis. More complex carrier geometries (biochips, nanotechnology) are also regarded as microarrays in the sense of this embodiment, and controlled chemical reactions are carried out on their surfaces.
  • the molecules attached to the surface are referred to as the target (target library).
  • the mixture of substances to be examined, which is brought into contact with the array is referred to as a (complex) sample.
  • the sample and array are brought into contact under hybridization conditions, and non-binding sample molecules are removed again.
  • the number of sample molecules remaining on the support forms a hybridization profile and allows the molecular composition of the sample to be determined [1,2].
  • the molecules remaining on the array are identified by suitable labeling (for example: fluorescent or radioactive labeling) and measured using appropriate detection methods (for example fluorescence or laser scanners).
  • suitable labeling for example: fluorescent or radioactive labeling
  • detection methods for example fluorescence or laser scanners.
  • the object of the invention is to eliminate the disadvantages of the previously known methods and to develop a method which enables the parallel quantitative measurement of molecular amounts in complex mixtures.
  • a complex mixture of reference molecules is added to the sample (e.g. DNA, RNA or polypeptides) and applied to an array which, in addition to the detector molecules matching the sample, also contains target molecules to which the reference molecules bind in a manner known per se.
  • the reference molecules are selected so that they have similar binding characteristics as the sample molecules, but do not interact with each other, with the sample molecules or the associated detector molecules.
  • An example of this is that a mixture of plant-specific Arabidopsis cDNA clones is added for the analysis of a complex sample from mouse cDNA. The GC content of these clones corresponds to that of the mouse clones but does not interact with them due to their sequence.
  • concentrations of the reference molecules in the reference mixture are preferably selected so that they are an image of the sample molecules in the sample mixture.
  • the measurement is carried out in such a way that the reference molecules are applied to the array several times and the measurement error is determined from this multiple measurement.
  • the target molecules for the reference sample are applied to the entire array in an orderly fashion.
  • the method according to the invention can also be used on other microstructured supports such as e.g. Chips or nanotiter plates are carried out.
  • Optical, electrical or mechanical methods are used to detect the bound samples and reference molecules or the markers attached to them.
  • the array can significantly improve the quantitative measurement of the concentration of the sample molecules. This also ensures that different measurements can be compared.
  • the method according to the invention can be used for all types of molecules which enter into stoichiometric interactions (form key-lock pairs), in particular for RNA, PNA, DNA and proteins. It consists of the following steps:
  • the method can be used in conjunction with all possible technologies for determining the amount of molecules. In particular, it is independent of the exact technology used to label the molecules, such as radioactive labeling or fluorescent single or multi-color labeling.
  • a suitable selection must ensure that the molecules used as reference interact as little as possible with the molecules from the target library.
  • the reference molecules can be both known clones / proteins, for example from existing molecule libraries, and new, suitably designed molecules. In their binding properties, the reference molecules should reflect the chemical characteristics of the sample (eg in length distribution or GC content) and at the same time no interaction (such as background hybridization or cross-hybridization) with the samples and enter into the target molecules.
  • the sequences of all expected DNA molecules in the sample and all spotted DNA molecules in the target should be known. This is the safest way to avoid an interfering interaction with the sample or the target molecules.
  • Reference molecules should be selected to carry out a yeast hybridization experiment.
  • the genome of the archebacterium Archeoglobus fulgidus and Methanococcus janascii are available as a search library.
  • a similarity threshold e.g. an e-value for BLAST
  • a set of molecular candidates with sufficiently low similarity to the yeast genome is selected.
  • a set of non-homologous molecules is selected from these candidates (for example using the method described in [6]). If necessary, a further selection based on the criteria of the molecular composition (length, GC content) can be carried out.
  • a similarity threshold e.g. an e-value for BLAST
  • oligonucleotides In the case of oligonucleotides, a de novo synthesis of non-organism molecules is obvious.
  • Favorable molecular candidates of length L L nucleotides
  • L nucleotides can be found by selecting sparsely populated regions on the generalized hypercube [8] of the L nucleotides.
  • a simple numerical method provides the M oligonucleotides and all L nucleotides that appear in the genome of the organism to be examined with repulsive charges and searches for a configuration of minimal energy in the M * L-dimensional configuration space of the M L nucleotides by deterministic or stochastic iteration.
  • the amounts of the different reference molecules in the sample are to be selected so that they cover the entire range of the sample amounts to be examined.
  • a gradation of the different amounts in the form of a dilution series has proven itself.
  • a lower limit of the dilution series (lowest molecular concentration) is typically specified by the detector sensitivity.
  • the amount of normalization molecules in the target should correspond to the typical amount of target library molecules applied to the array. If this amount is highly variable, a dilution series should also be made for the target molecules. The procedure is explained in more detail in section 5, example 2.
  • the selected molecular data set is verified in a preliminary examination in which the reference molecules are brought into contact with the array individually and together, but without adding the complex sample: there should be no interactions with the target clones intended for sample measurement.
  • the sample is placed on the array without the addition of reference molecules: there should be no interactions with the target clones determined as reference.
  • a (generally non-linear) calibration curve can be determined from the signal strengths of the reference molecules by optimizing suitable model parameters or suitable interpolation methods.
  • the amount of sample can be determined from the signal strength by analytical or numerical inversion of this calibration curve. If there are free parameters that influence the binding behavior of the molecules, such as the GC content in DNA molecules, a whole host of transfer functions must be determined. In the case of spatially inhomogeneous arrays, the determination of several calibration curves is also necessary.
  • the reference molecules are diluted to different concentrations, for example in such a way that their concentration c decreases in ten steps, each by a factor of two (c 1 # ..., C ⁇ 0 ). This means that a measuring range of three decades can be controlled. The exact choice of dilution levels depends on the dynamic measuring range and the required experimental accuracy.
  • the normalization molecules are added to the complex sample (spiked) and the complement molecules are attached to the carrier in a number corresponding to the other target molecules.
  • T (c) the transmission function
  • this function is defined by linear interpolation of the measurement points; in general, a model function will be adapted to the measurement data (see Fig. 1).
  • the quantification function Q (s) T "1 (s) can be determined.
  • the corresponding curve When converting the signal intensity into the molecule concentration, the corresponding curve must then be selected. If, for example, you want to determine the molecule concentration from the measured signal for a molecule with a GC content of 30%, the lowest quantification curve is used. If the influence of the GC content is known from general considerations, the one-dimensional dilution series discussed in Example 1 is sufficient. The GC content then appears explicitly as an additional parameter in the transmission function. Is an analytical relationship is not known, has for a representative parameter range GC m i n ..GC max of influence are examined for the transmission function.
  • a two-dimensional dilution series is required. Analogous to Example 1, dilution series are formed. If the sample concentration c p is to be measured at n levels and the target concentration c t at m levels, it makes sense to choose mxn different standardization molecules in order to ensure the independence of the dilution experiments. Now you create a dilution matrix of n rows and m columns: Each matrix entry corresponds to a complementary pair of samples and target molecule.
  • the concentration of the sample molecules c p is reduced from row to row in accordance with the sample dilution series.
  • the concentration of the target molecules c t is reduced from column to column according to the sample dilution series.
  • the measurement quality is determined by the reproducibility of measurements.
  • the reproducibility is given by the expected measurement error in the case of a repeat measurement (mean fluctuation square).
  • the typical measurement error at must be determined depending on the given signal strength.
  • a reference point for the measurement error can already be obtained by repeating the standardization series.
  • the effective measurement accuracy in the entire measuring range can be derived (see Fig. 1 and Fig. 2).
  • real quality control requires the entire experiment to be repeated.
  • the multiple measurement in turn allows the effective fluctuation range to be determined both for the individual measurement and for a given intensity range using the inverse function.
  • Fig. 1 Construction of the calibration curve (transmission function T) based on six repetitions of a dilution series.
  • Fig. 2 Reconstruction of the molecular concentrations using the quantification function Q. The six repetitions of the dilution series serve to estimate the expected measurement error.
  • Fig. 3 If the observed signal intensity is strongly dependent on a parameter, such as the GC content, a host of quantification functions must be determined.

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  • Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
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  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour mesurer quantitativement des quantités de molécules dans des mélanges complexes à l'aide de jeux ordonnés de microéchantillons et d'autres supports analogues. Les domaines d'application de la présente invention sont la biologie moléculaire, la médecine et l'industrie pharmaceutique.
PCT/EP2001/006009 2000-05-24 2001-05-25 Mesure quantitative de quantites de molecules dans des melanges complexes Ceased WO2001090407A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10025384A DE10025384A1 (de) 2000-05-24 2000-05-24 Quantitative Messung von Molekülmengen im komplexen Gemischen
DE10025384.9 2000-05-24

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WO2001090407A1 true WO2001090407A1 (fr) 2001-11-29

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DE102007055386B4 (de) * 2007-11-20 2015-07-16 Boehringer Ingelheim Vetmedica Gmbh Verfahren zur Kalibrierung eines Sensorelements

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5620901A (en) * 1993-03-30 1997-04-15 Terrapin Technologies, Inc. Determination of concentration by affinity titration
WO1999051772A1 (fr) * 1998-04-07 1999-10-14 Incyte Pharmaceuticals, Inc. Dosages quantitatifs par hybridation de micro-arrangements
US6004775A (en) * 1990-08-03 1999-12-21 The Salk Institute For Biological Studies DNA encoding IGFBP-4
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004775A (en) * 1990-08-03 1999-12-21 The Salk Institute For Biological Studies DNA encoding IGFBP-4
US5620901A (en) * 1993-03-30 1997-04-15 Terrapin Technologies, Inc. Determination of concentration by affinity titration
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
WO1999051772A1 (fr) * 1998-04-07 1999-10-14 Incyte Pharmaceuticals, Inc. Dosages quantitatifs par hybridation de micro-arrangements

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EICKHOFF B ET AL: "NORMALIZATION OF ARRAY HYBRIDIZATION EXPERIMENTS IN DIFFERENTIAL GENE EXPRESSION ANALYSIS", NUCLEIC ACIDS RESEARCH, IRL PRESS LTD., OXFORD, GB, vol. 27, no. 22, 15 November 1999 (1999-11-15), pages E33I - E33III, XP001018017, ISSN: 0305-1048 *
EICKHOFF H ET AL: "ROBOTIC EQUIPMENT AND MICROSYSTEM TECHNOLOGY IN BIOLOGICAL RESEARCH", BIOMETHODS, BIRKHAEUSER, BASEL, CH, vol. 10, 1999, pages 17 - 30, XP001018280, ISSN: 1018-6255 *
EICKHOFF H, ET AL: "Tissue Gene Expression Analysis Using Arrayed Normalized cDna Libraries", GENOME RESEARCH, vol. 10, 10 August 2000 (2000-08-10), pages 1230 - 1240, XP002180561 *
SCHUCHHARDT J ET AL: "NORMALIZATION STRATEGIES FOR CDNA MICROARRAYS", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 28, no. 10, 15 May 2000 (2000-05-15), pages E47I - E47V, XP000982625, ISSN: 0305-1048 *

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