WO2004024949A2 - Procede de detection rapide de mutations et de polymorphismes nucleotidiques par chimiometrie - Google Patents

Procede de detection rapide de mutations et de polymorphismes nucleotidiques par chimiometrie Download PDF

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WO2004024949A2
WO2004024949A2 PCT/DK2003/000594 DK0300594W WO2004024949A2 WO 2004024949 A2 WO2004024949 A2 WO 2004024949A2 DK 0300594 W DK0300594 W DK 0300594W WO 2004024949 A2 WO2004024949 A2 WO 2004024949A2
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probe
nucleotides
polynucleotide
target
target polynucleotide
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WO2004024949A3 (fr
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Mogens Fenger
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Hvidovre Hospital
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Publication of WO2004024949A3 publication Critical patent/WO2004024949A3/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
    • G06N20/00—Machine learning
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
    • G06N3/00—Computing arrangements based on biological models
    • G06N3/02—Neural networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Definitions

  • the present invention relates to the field of molecular biology and more specifically to methods and kits for detection of hybrid polynucleotides between a target and a labelled probe polynucleotide.
  • the methods and kits may be used for the determination of mutations and polymorphisms in samples containing target polynucleotides. All patent and non-patent references cited in the application are hereby incorporated by reference in their entirety.
  • the DNA to be assayed is denatured and bound to a solid support, e.g. beads, nitrocellulose or nylon, and then incubated with a probe sequence, complementary to the target, which contains a label, for example 32 P, or one half of an affinity pair, for example biotin. After incubation and washing of the solid phase, it is then developed to give a signal, by autoradiography for 32 P, or in the case of biotin, typically by the addition of an avidin-enzyme conjugate, further washing and then substrate.
  • sandwich assays are known, whereby a capture nucleic acid probe, of sequence complementary to the target, is bound to a convenient solid support.
  • a labelled probe is then allowed to hybridise to another part of the target sequence, and after washing steps, the label is developed as appropriate.
  • the "sandwich" is formed in solution with two probes, one containing the label and the other containing one half of an affinity pair. After hybridisation, the solution is then contacted with the solid phase to which the other half of the affinity pair is bound. Washing and signal development then takes place. All these methods and their variants require numerous handling steps, incubations, washings, and the like which result in processes which are both laborious and time-consuming.
  • Nucleic acid sequences may be amplified prior to hybridisation to increase the number of target nucleic acids.
  • Methods for detecting amplified nucleic acid sequences for example DNA sequences generated by PCR, are as outlined above. Alternatively, they can be analysed by gel electrophoresis, and the existence of a band of a given molecular weight is taken as evidence of the presence of the sequence in the original DNA sample, as defined by the specific primers used in the amplification reaction. Again such methods are relatively time-consuming and laborious.
  • hybridisation assays include some sort of signal amplification, which may comprise binding of more labelled probe to the same target nucleic acid or the use of an enzyme based system, wherein the amount of signal produced per probe can be increased by letting the enzymes convert more substrate to detectable product.
  • signal amplification involves the use of nucleotide amplifiers as described in US 5,635,352 and US 5,124,246 (assigned to Chiron).
  • Signal detection in most of the known methods is relatively simple in that only one signal is determined and this is done at one wavelength only. Most often, the determination is a qualitative determination of whether there is a significant signal or not.
  • Polymorphisms and mutations are ubiquitous in the genome. The distinction between polymorphisms and mutations lies in the frequency of alleles in a gene, where a mutation is defined if one allele amounts to more than 99% of the alleles, and polymorphisms accounts for all other situations. Both polymorphisms and mutations can be single nucleotide substitutions, deletions, insertions or rearrangements. As an consequence of the Human Genome Project interest has been focussed particularly on single nucleotide polymorphisms (SNP) for various reasons including their usage in genome scanning for diseases-related genes. More than 2 million SNP's has been detected, some but not all of which are directly related to diseases.
  • SNP single nucleotide polymorphisms
  • DNA sequencing (Sanger et al. (1977) Nature, 265: 678-695; Maxam and Gilbert (1977) Proc Natl Acad Sci USA, 74: 560- 564); the single strand conformation polymorphism (SSCP) method (Orita et al. (1989) Genomics, 5:874-879); the denaturing gradient gel electrophoresis (DGGE) method (Fisher and Lerman. (1993) Proc. Natl. Acad. Sci.
  • SSCP single strand conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • Mutations can be detected by specific hybridisation of oligonucleotides to the nucleic acid to be analysed.
  • US 6,022,686 (Zeneca Ltd) describes the simultaneous use of fluorescent probes for the detection of mutations by liquid phase hybridisation and fluorescence polarisation. Since the detection occurs by hybridisation of nucleic acid sequences in homogeneous solution, the method allows detection of probe specific sequences from both chromosomes.
  • Variations in DNA-sequences may also be detected by hybridisation in a system consisting of a solid and a liquid phase (two-phase systems).
  • One particular useful approach is the so-called “sandwich” hybridisation described in US 4,486,539 (Orioon Corp. Ltd.) .
  • Many haplotypes are described by single base polymorphisms, but US 4,486,539 do not provide methods to detect single base polymorphisms. The same is true for the majority of the methods for detection of hybrid nucleotides described above. For most of these methods, it is very difficult to design conditions under which one probe is capable of distinguishing between target nucleotides differing in one position only.
  • spectral data a spectrum is understood as a spectrum covering a range of wavelengths of electromagnetic radiation, including vacuum ultraviolet, ultraviolet, visible, near infrared, infrared, far infrared light, microwaves, and radiowaves.
  • the spectral data must be in a format so that they can be exposed to multivariate analysis.
  • performance of mass spectrometry is also regarded as recording a spectrum, although in this case no electromagnetic radiation is emitted or recorded. Also in this case no detectable label is used.
  • a detectable label refers to a chemical moiety capable of emitting, absorbing or scattering electromagnetic radiation. More preferably, a label refers to chemical moieties capable of emitting, absorbing, or scattering phosphorescence, luminescence, or fluorescence.
  • Hybrid polynucleotide a hybrid between two polynucleotides, wherein the association between the two polynucleotides is stronger than the association between one of the polynucleotides and water.
  • the methods according to the present invention provide simple analytical techniques compared to the prior art methods for determination of hybrid polynucleotides. This will render the analytical techniques according the present invention available for laboratories without special molecular biological expertise. The methods are simple and cheap both with regard to equipment and use.
  • the invention will also solve some of the problems of the research into diseases having a genetic factor.
  • the problem is that many of the important diseases, such as diabetes, artheresclerotic and psychiatric diseases are of polygenic nature meaning that the interaction between several genes leads to a predisposition or directly leads to these diseases. Elucidation of the genetic component of these diseases requires a large number of patients and a significant analytical effort.
  • the present invention partly reduces the costs associated with these analyses and partly accelerates the investigations. It is expected that the present invention will partly replace the main part of the gene- diagnostic methods based on electrophoresis and sequencing. It is likewise expected to supplement the so-called chip based gene-diagnostic methods.
  • the invention relates to a method for establishing whether at least one target polynucleotide is present in a sample, comprising the steps of
  • the at least partly complementary probe is capable of hybridising to the sub-sequence of the target polynucleotide.
  • the analysis of the spectral data can distinguish for each of the at least one probe whether the probe is part of the at least one hybrid polynucleotide or not part of the at least one hybrid polynucleotide.
  • the analysis of the spectral data can distinguish for each of the at least one probe, when the probe is part of the at least one hybrid polynucleotide, whether or not there is a mismatch between the probe and the sub-sequence of the at least one target polynucleotide.
  • the spectral data are analysed using multivariate analysis.
  • the invention relates to a method for identifying a hybrid polynucleotide comprising at least partly complementary nucleotide strands, said method comprising the steps of
  • polynucleotide probe comprises at least one detectable label
  • hybrid polynucleotide comprising at least one target polynucleotide and at least one polynucleotide probe
  • spectral data when at least one oligonucleotide probe forms part of the hybrid polynucleotide are different from the spectral data when at least one oligonucleotide probe is not part of the hybrid polynucleotide.
  • spectral data instead of only data from one wavelength it is possible to determine the presence or absence of a particular hybrid polynucleotide even in the presence of the unbound polynucleotide probe. It is thus possible to record a spectrum from an environment comprising both the hybrid and unbound probe and by comparing that spectrum with spectra of the unbound probe and the hybrid determine whether a hybrid has formed or not. It is also possible to distinguish the spectrum from a hybrid between target and probe with a given number of mismatches from the spectrum of a hybrid between target and probe with a higher number of mismatches. It is thus e.g. possible to distinguish the spectrum from a hybrid where there is a stretch of 100% complementarity between probe and target (i.e.
  • the analytical method is fast, easy to use and can be completely automated. It is easy to include various control steps in the method and thereby ensure that the detected results can be verified.
  • the invention relates to a method for detecting a hybrid between a target polynucleotide and a probe oligonucleotide comprising
  • polynucleotide probe comprises at least one detectable label
  • hybrid polynucleotide comprising at least one target polynucleotide and at least one polynucleotide probe
  • the invention relates to a method for detecting a mutation or polymorphism, said method comprising the steps of
  • polynucleotide probe comprises at least one detectable label
  • a hybrid polynucleotide comprising at least one target polynucleotide and at least one polynucleotide probe
  • the invention relates to a kit for detection of mutations or polymorphism comprising
  • At least one oligonucleotide probe capable of hybridising to a preselected region of a target polynucleotide, the polynucleotide probe further comprising at least one detectable label,
  • the instructions may be in the form of calibration data relating to the specific assay to which the kit relates, so that the user of the kit does not have to carry out extensive analyses to determine the grouping of spectra in multivariate analysis.
  • the invention relates to a system for establishing whether at least one target polynucleotide is present in a sample, comprising i) at least one polynucleotide probe being at least complementary to a target polynucleotide, the probe comprising a detectable label, ii) a sample chamber from which electromagnetic radiation can be recorded, iii) a source of spectrally resolved electromagnetic radiation, iv) means for sensing and recording a spectrum of electromagnetic radiation from the sample chamber, and v) a computer unit for storing spectral data of electromagnetic radiation and having instructions to treat the recorded spectral data using multivariate analysis.
  • the invention relates to a system for detection of a hybrid polynucleotide comprising i) at least one oligonucleotide probe being at least partly complementary to a target polynucleotide, the probe comprising a detectable label, ii) a sample chamber from which electromagnetic radiation can be recorded, iii) a source of spectrally resolved electromagnetic radiation, iv) means for sensing and recording a spectrum of electromagnetic radiation from the sample chamber, and v) a computer unit for storing spectral data of electromagnetic radiation and having instructions to treat the recorded spectral data using multivariate analysis.
  • the system is adapted for performance of the methods according to the present invention and provides a system which allows high throughput screening of samples.
  • Figure 1 The underlying idea of PCA modelling is to replace a complex multidimensional data set by a simpler version involving fewer dimensions, but still fitting the original data closely enough to be considered a good approximation.
  • a 3- D data swarm is shown.
  • the new axes (the principal components PC1 and PC2) are placed in the directions of the largest variances. In this example 3 dimensions are reduced to 2.
  • PC 1 is the first principal component, which is most important in explaining the difference between the samples.
  • PC 2 is the next important, PC 3 the third and so on.
  • FIG. 1 A PC1/PC2 loading-plot.
  • Figure 4 Nucleotide section of the Apo3611 region. Complementary wildtype and mutant oligonucleotide probe.
  • FIG. 5 PCA scores plot based on emission spectra obtained from the room temperature hybridisations of Cy5 labelled oligonucleotides (A: Cy5 labelled wildtype oligonucleotide interrogating the wildtype nucleotide at the 5'end, B: Cy5 labelled mutant oligonucleotide interrogating the wildtype nucleotide at the 5'end, C: Cy5 labelled wildtype oligonucleotide interrogating the wild type nucleotide in the central part of the probe or D: Cy5 labelled mutant oligonucleotide interrogating the wildtype nucleotide in the central part of the probe) in the presence or absence of wildtype target DNA (W1).
  • A Cy5 labelled wildtype oligonucleotide interrogating the wildtype nucleotide at the 5'end
  • B Cy5 labelled mutant oligonucleotide interrogating the wildtype nucleotide at the 5'end
  • C Cy5
  • the spectra were either obtained from the oligonucleotides alone or in combination with target (denoted X_W).
  • the first prefix (1 or 2) gives the replicate number, whereas the second prefix represent two fluorescence measurements on that same replicate solution.
  • FIG. 7 PCA scores plot based on emission spectra obtained from 30°C hybridisations of Cy5 labelled oligonucleotides (A, B, C or D) in the presence of wildtype target DNA (W1 ). Data points are the same as for Figure 6 but spectra of fluorescent oligonucleotides without targets have been left out. The prefix (1 -3) gives the replicate number. PCA scores plot from experiment 2.
  • Figure 8 PCA scores plot based on excitation spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotides (A, B, C or D) in the presence or absence of wildtype target DNA (W1). The prefix (1-3) gives the replicate number.
  • FIG. 9 PCA scores plot based on excitation spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotides (A, B, C or D) in the presence of wildtype target DNA (W1). Data points are the same as for Figure 8 but spectra of fluorescent oligonucleotides without targets have been left out. The prefix (1-3) gives the replicate number. PCA score plot from experiment 2.
  • FIG. 12 PCA scores plot based on excitation spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotide A in the presence or absence of wildtype target DNA (A-W2), mutant target (A-M) or both DNA targets simultaneously (A-W2-M, note concentrations: 0,125 or 0,25 ⁇ M).
  • A-W2-M-2_2 is not included while it was considered as an outlier.
  • the prefix (-1 or - 2) represent two different concentrations of the targets, and the prefix (1-3) give the replicate number. Data are from experiment 4. An outlier has been removed from the data analysis.
  • Figure 13 PCA scores plot based on emission spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotide A in the presence or absence of wildtype target DNA (A-W2), mutant target (A-M) or both DNA targets simultaneously (A-W2-M).
  • the prefix (-1 or-2) represent two different concentrations of the targets, and the prefix (1-3) give the replicate number. Data are from experiment 4.
  • Figure 14 PCA scores plot based on Cy5 excitation spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotides A or B and with or without Cy3 labelled oligonucleotide F in the presence or absence of wildtype target W3. The prefix (1 -3) gives the replicate number. Data are from experiment 5.
  • Figure 15 PCA scores plot based on emission spectra obtained from 30 °C hybridisations of Cy5 labelled oligonucleotides A or B and with or without Cy3 labelled oligonucleotide F in the presence or absence of wildtype target W3. The prefix (1 -3) gives the replicate number. Data are from experiment 5.
  • Figure 16. Illustrates fluorescence spectra of a target polynucleotide (W1 of Figure 4) alone and the PCR buffer used for amplification alone.
  • Figure 17 Shows on the left fluorescence spectra of two different probes labelled with Cy5 (A and B from Figure 4). On the right is shown the two spectra recorded from samples with both the probe and the target polynucleotide. The interaction between target and probe changes the spectrum.
  • FIG. 18 Shows on the left fluorescence spectra of two different probes labelled with Cy5 (C and D from Figure 4). On the right is shown the two spectra recorded from samples with both the probe and the target polynucleotide. The interaction between target and probe changes the spectrum.
  • FIG. 19 PCA-plot of dsDNA after hybridization to Cy5-labelled probe.
  • ssDNA-probe complexes are included for illustrative purposes (AM and
  • AM-Rm probe complexed with dsDNA mutant
  • AW2-R probe complexed with dsDNA wildtype
  • AW2-R- probe complexed with dsDNA wildtype, but the denaturation step is omitted
  • A probe alone.
  • Figure 20 PCA-plot of Poly2-dsDNA after hybridization to Cy5-labelled probe.
  • ssDNA-probe complexes are included for illustrative purposes (dWP- WA and dWP-MA), but are usually not included in the analysis.
  • dWP-WA-WS probe complexed with dsDNA wildtype Poly2
  • dWP-MA-MS probe complexed with dsDNA mutant Poly2.
  • Figure 16 shows a fluorescence spectrum of the target DNA alone (top) and the PCR buffer alone (bottom). The differently shaded curves represent different replicate samples.
  • Figure 17 on the left is shown spectra of the probes (A and B as described in the examples and Figure 4). Note the difference in the scale of the Y axis between Figure 16 and Figure 17/18. The buffer and target alone do not contribute significantly to the spectrum of Figures 17/18.
  • probes A and B the mutant nucleotide is placed terminally and the label is linked directly to the mutant nucleotide.
  • probe A which is 100 % complementary to a sequence in the target polynucleotide interacts differently with this than probe B, which differs in one position.
  • probe B which differs in one position.
  • the recorded spectrum is changed slightly. In this particular case the difference is just visible in the spectrum, but in order to make full use of the information in the spectra multivariate analysis is used.
  • These changes can be analysed using multivariate analysis. It can also be seen that the spectrum of probe B, which differs from probe A at one position (see Figure 4) is not changed to the same degree by interaction with the target polynucleotide.
  • FIG 18 a further example is illustrated.
  • the probes (C and D) differ from probes A and B in that the mutant nucleotide is placed centrally in the probe (see Figure 4).
  • the spectra to the right of Figure 18 are recorded from samples with both the probe and the target polynucleotide, which is 100 % complementary to probe C and differs from probe D at one (terminal) position. Again the association between target and probe changes the spectrum so that the presence of a target wildtype can be distinguished form a target mutant polynucleotide.
  • Chemometrics in analogy with biometrics and econometrics. Chemometrics is heavily dependent on the use of different kinds of mathematical models - high information models, ad-hoc models, and analogy models. The task demands knowledge of statistics, numerical analysis, operation analysis, etc., and applied mathematics. However, as in all applied branches of science, the difficult and interesting problems are defined by the applications. There is a tendency to make fewer experiments and measure more and more data in each of them. This trend is seen everywhere, ranging from physics with high costs for accelerators and other equipment to biomedical research where ethical and regulatory aspects provide a strong incentive for fewer experiments.
  • Multivariate data analysis/chemometrics convert any more or less structured and complicated sets of data into a few meaningful plots that display the information hidden in the data structures in a way that is easy to understand.
  • PCA Principal Component Analysis
  • the principle of the analysis is to arrive at a simplified set of data (data reduction) whilst retaining as much information as possible. This means that an attempt is made at trying to determine which variables are essential to the total variation in the set of data and to determine whether some of the variables provide the same information (i.e. separate the samples in the same way). By doing this, an attempt is made at uncovering the underlying structure of the data material.
  • PCs The principal components
  • the principal components are extracted one after the other by mapping where the greatest total variation in the data set is to be found.
  • the first principal component is placed where the greatest distances between the samples are to be found, and the analysis provides a percentage value of how much of the total variation is explained by looking in the direction of the first principal component.
  • the next principal components are found as directions/axes on which the rest of the variation in the set of data can be found. Gradually, as more and more principal components are found one will acquire more and more descriptions of the
  • SUBSTITUTE SHEET RULE 26 differences between the samples with respect to the given variables. Usually, the data set includes noise and since this noise is not used to describe the differences between samples there is a limit to how much variation one can describe.
  • Figure 1 visualises how the 2 first principal components (PC1 and PC2) are found in a 3 dimensional plot. Only data of 3 variables are available, so the linear behaviour is immediately recognised by plotting the objects in the 3-D variable space. When more variables are present (like in spectroscopy where each row of a data matrix is a spectrum of perhaps several hundred of wavelengths), this procedure is of course not feasible. Identification of this type of linear behaviour in a space with several hundred dimensions of course cannot be achieved by visual inspection.
  • PCA with its powerful projection characteristics, helps to discover the hidden data structures.
  • Pre-processing is used to ensure that the raw data have a distribution that is optimal for the analysis. Background effects, measurements with various units, different variance of the variables, etc. makes it difficult to extract the meaningful information. Pre-processing reduces the noise introduced by such effects.
  • Pre-processing may contain the operations of centering, weighting and transformations. In the examples transformations have not been used.
  • Transformations include logarithming, smoothing, deriving, normalisation, scatter correction.
  • the score plot shows the clustering of different samples. Samples that lie close to each other in the score plot (sample2 and sample ⁇ ) are similar with respect to the measured variables. On the other hand, the samples that lie diagonally opposite each other in the plot will be very different from each other (sample3 vs. sample ⁇ or sample2 and sample6 vs. samplel). Additionally, the samples, which lie close to zero in the plot (sample4), are near to the average with regard to the variables measured.
  • the loading plot ( Figure 3) is a visualisation of the measured variables that can be attributed the greatest weight when separating the samples.
  • the variables that lie far away from the axes, either in a positive or a negative direction (PARAM.2, PARAM.3, PARAM.4, PARAM.5, PARAM.6 and PARAM.7) will be of greatest importance when separating the samples.
  • Each of the axes in the plot represent a principal component which means that they describe a certain part of the differences in the set of figures, and this is usually depicted in the corner of the plot.
  • the variables that lie far out on the axes with high percentages are most important. That is, these variables are most prominent in explaining the differences between samples.
  • an outlier is an observation (outlying sample) or variable (outlying variable) which is abnormal compared to the major part of the data.
  • Extreme points are not necessarily outliers; outliers are points that apparently do not belong to the same population as the others, or that are badly described by a model. Outliers should be investigated before they are removed from a model, as an apparently outlier may be due to an error in the data.
  • multivariate analysis comprises general multivariate analysis, principal component analysis and extensions of this, exploratory and confirmatory factor analysis in its various forms, Cluster and latent class analysis including scaled latent class analysis, structural equation analysis, Fixed mixture analysis and combinations hereof.
  • the spectral data may be used for training a neural network.
  • the neural network Once the neural network has been trained with a sufficiently high number of known spectra, the neural network is capable of grouping a spectrum from an unknown sample to the correct group, e.g. wildtype, mutant, non-interacting sequence, homozygote heterozygote.
  • Spectroscopic techniques form the largest and most important single group of techniques used in analytical chemistry, and provide a wide ranges of quantitative and qualitative information. All spectroscopic techniques depend on the emission or absorption of electromagnetic radiation characteristic of certain energy changes within an atomic or molecular system. The energy changes are associated with a complex series of discrete or quantised energy levels in which atoms and molecules are considered to exist.
  • the spectroscopy comprises ultraviolet spectrometry, visible spectrometry or infrared spectrometry. These spectroscopic methods can all be used in conjunction with numerous known labels and the equipment used for recording the spectra is standard laboratory equipment and thus generally available.
  • Recording a spectrum under normal conditions comprises recording a value (absorption, extinction, emission etc) for a number of discrete wavelengths, because spectroscopes normally do not record continuous spectra.
  • the recording of a spectrum comprises recording at as many discrete wavelengths as possible or recording a continuous spectrum if possible.
  • recording spectral data preferably comprises detection of signal for at least 10 discrete wavelengths, more preferably at least 20 discrete wavelengths, more preferably at least 50 discrete wavelengths, more preferably at least 100 discrete wavelengths, more preferably at least 200 discrete wavelengths, more preferably at least 250 discrete wavelengths, more preferably at least 300 discrete wavelengths, more preferably at least 400 discrete wavelengths, more preferably at least 500 discrete wavelengths, more preferably at least 600 discrete wavelengths, more preferably at least 750 discrete wavelengths, more preferably at least 1000 discrete wavelengths, such as at least 1250 discrete wavelengths, for example at least 1500 discrete wavelengths, such as at least 2000 discrete wavelengths.
  • the distance between the different wavelengths preferably is as low as possible. This increases the possibility of resolving the spectra into different groups. Accordingly, the distance between the discrete wavelengths preferably is 10 nm or less, more preferably 5 nm or less, more preferably 3 nm or less, more preferably 2 nm or less, more preferably 1 nm or less, more preferably 0.8 nm or less, more preferably 0.75 nm or less, more preferably 0.7 nm or less, more preferably 0.6 nm or less, more preferably 0.5 nm or less, more preferably 0.25 nm or less, more preferably 0.1 nm or less, more preferably 0.05 nm or less, more preferably 0.01 nm or less.
  • the spectral data recorded comprises a fluorescence spectrum between 180 and 950 nm.
  • a fluorescence spectrum may be an excitation spectrum or an emission spectrum, or both, depending on the level of reduction of variance not related to the interaction of target and non-target. To the extent that these non-interactive variances can be eliminated e.g. by clustering the spectre both emission and excitation spectre alone or in combination can be of analytical value.
  • the method preferably further comprises recording of spectral data from the polynucleotide probe alone. After having established this difference, it is expected that it is not always necessary to record this spectrum, although it may in some instances be useful as a calibration for day-to-day variation.
  • the method may further comprise recording spectral data from the hybrid polynucleotide and from a polynucleotide probe alone and/or, from a non-hybridising polynucleotide probe contacted by the target polynucleotide, and/or from a polynucleotide probe contacted with a non-hybridising polynucleotide sequence. All of these spectra, which may be regarded as "controls" serve the purpose of calibrating the method.
  • Mass spectrometry is a technique for characterising molecules according to the manner in which they fragment when bombarded with high-energy electrons, and for elemental analysis at trace levels. It is not strictly speaking a spectrometric method as electromagnetic radiation is neither absorbed nor emitted. However, the data obtained are in a spectral form in that the relative abundance of mass fragments from a sample is recorded as a series of lines or peaks. The bombardment process produces many fragments carrying a charge, and this facilitates their separation and detection by electrical and magnetic means. Spectra must be recorded under conditions of high vacuum (10 "4 to 10 "6 Nm "2 ) to prevent loss of the charged fragments by collision with molecules of atmospheric gases or swamping of the sample spectrum. In the case of using mass spectrometry for recording the spectrum, it is contemplated that it may not always be necessary to include a label in the polynucleotide probe, since the association between probe and target alone is enough to create differences in the recorded spectrum.
  • label means a chemical moiety which is coupled to a nucleic acid of a polynucleotide probe (possibly via a molecular linker) and which can be used as a signal source for electromagnetic radiation or as a source of interaction with electromagnetic radiation supplied to the label.
  • the polynucleotide probes may be labelled by a number of methods well known in the art. Conveniently, polynucleotide probes may be labelled during their solid-phase synthesis using any of the many commercially available phosphoramidite reagents for 5' labelling. Illustrative examples of oligonucleotide labelling procedures may be found in US
  • a preferred label according to the invention has a fairly complex spectrum, which when resolved at short wavelength distances produces more than one local maximum in addition to the global maximum.
  • the recorded spectrum When interacting with one target polynucleotide the recorded spectrum preferably changes at more than one wavelength. Thereby more data can be accumulated and used for the multivariate analysis.
  • a fluorescent, phosphorescent or luminescent label is preferred because it provides a strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. More preferably the label comprises a fluorescent label.
  • a particular fluorescent label has a characteristic excitation and emission spectrum which allows the simultaneous detection of several different fluorescent labelled molecules if the labels are selected appropriately.
  • a large number of different useful fluorescent labels are given in the art and may be selected from the group comprising, but not limited to: Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (trademarks for Biological Detection Systems, Inc.), fluorescein, acridin, acridin orange, Hoechst 33258, Rhodamine, furthermore: Rhodamine Green, Tetramethylrhodamine, Texas Red, Cascade Blue, Oregon Green, Alexa Fluor (trademarks for Molecular Probes, Inc.), 7-nitrobenzo-2-oxa-1 -diazole (NBD), pyrene and Europium, Ruthenium, Samarium, and other rare earth metals, bimane, ethidium, europium (III) citrate, La Jolla blue, methylcoumarin, nitrobenzofuran, pyrene butyrate, rhodamine, and terbium chelate. More specialised fluorochromes are listed in Table 2 along with their
  • the label comprises a Cy5 dye.
  • a quencher is added to the sample with the hybrid between target and probe. This may be preferable in certain cases in order to quench parts of the spectrum, which only contributes to noise. This would be most appropriate when non-relevant variance (noise) cannot be eliminated by multivariate analysis. In some instances quenchers may act as modifiers to alter the general spectral characterstics.
  • quenchers depend on the specific part(s) of the spectrum to be quenched.
  • the quenching abilities of a number of different compounds are known in the art.
  • One particular example is the TAMRA quencher.
  • one or more probes or targets polynucleotide is immobilised to a solid surface.
  • the nature of the means for immobilisation and of the nature of the solid support is a matter of choice.
  • suitable supports and methods of attaching nucleotides to them are well known in the art and widely described in the literature.
  • supports in the form of microtiter wells, tubes, dipsticks, particles, fibers or capillaries may be used, made for example from agarose, cellulose, alginate, teflon, latex or polystyrene.
  • the support may comprise magnetic particles, which permits the ready separation of immobilised material by magnetic aggregation.
  • the solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups for the attachment of nucleotides. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups.
  • a polymer carrying one of such functional groups e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups.
  • US 4,654,267 describes the introduction of many such surface coatings.
  • the support may carry one member of an "affinity pair", such as avidin, while the polynucleotide is conjugated to the other member of the affinity pair in casu biotin.
  • affinity pair such as avidin
  • Representative specific binding affinity pairs are shown in Table 4.
  • streptavidin/biotin binding system is very commonly used in molecular biology, due to the relative ease with which biotin can be incorporated within nucleotide sequences, and indeed the commercial availability of biotin-labelled nucleotides, and thus biotin represents a preferred means for immobilisation.
  • an amplified DNA strand is labelled with a molecule, which is subsequently used to immobilise the labelled DNA strand to a solid surface.
  • the target polynucleotide may be labelled by a number of methods.
  • One convenient method to label DNA is to enclose one labelled amplification primer oligonucleotide in the amplification reaction mixture. During the amplification process the labelled oligonucleotide is built into the DNA fragment which become labelled.
  • oligonucleotides may also be labelled or coupled to chemoreactive groups comprising, but not limited to: sulfhyl, primary amine or phosphate.
  • chemoreactive groups comprising, but not limited to: sulfhyl, primary amine or phosphate.
  • SH-modified DNA may be immobilised on a gold surface (Steel et al. (2000) Biophys J 79:975-81) likewise 5'- phosphorylated DNA or 5'-aminated DNA may be immobilised by reaction with activated surfaces (Oroskar et al. (1996) Clin Chem 42:1547-55; Sjoroos et al (2001) Clin Chem 47:498-504).
  • oligonucleotides may also be labelled or coupled to photoreactive groups.
  • Acetophenone, benzophenone, anthraquinone, anthrone or anthrone-like modified DNA can for instance be activated by exposure to UV light and immobilised on a wide range of surfaces as described in European and US patents: EP0820483, US6033784 and US5858653.
  • photoreactive psoralens, coumarins, benzofurans and indols have been used for immobilisation of nucleic acids. An extensive discussion of immobilisation of nucleic acids can be found in WO 85/04674.
  • a target polynucleotide is defined as a polynucleotide with which at least one probe can form a hybrid polynucleotide.
  • RNA polynucleotides such as wherein the target polynucleotide comprises RNA, such as mRNA and/or rRNA and or tRNA.
  • RNAs including 5S, 5.5-5.8S, 16S, 18S, 23S, 25-28S rRNA can be used for identifying the taxon from which the RNA was isolated, since conserved sequences can be found for a number of taxons and for a number of species.
  • rRNA may for example be used for diagnosing an infectious disease caused by microorganisms, or for determining the amount and nature of contamination in food, feed and various water-supplies.
  • Another source of target polynucleotides are DNA. DNA may be used for determination of mutations and/or polymorphisms, but also in genotyping individuals or for determining the taxon, for forensic usage or for linkage studies.
  • the different sources of DNA include genomic DNA, organelle DNA, mitochondrial DNA, chloroplast DNA, cDNA, and environmental DNA.
  • the target polynucleotides may also comprises a synthetic polynucleotide sequence.
  • a restriction site in the target polynucleotide so that the sequences can be cloned into a vector afterwards. Mutations may also be included in the sequences during isolation and/or amplification, e.g. via so-called error-prone PCR.
  • synthetic sequences include but are not limited to the inclusion of various control polynucleotides in the hybridisation mixture, such as positive controls (wild-type, mutation, heterozygote), negative control (dummy DNA sequence).
  • the target polynucleotide comprises chemically or biologically modified nucleic acids, such as the modification comprising modification of cytosin by bisulphite.
  • chemically or biologically modified nucleic acids such as the modification comprising modification of cytosin by bisulphite.
  • modification comprising modification of cytosin by bisulphite.
  • This is the study of methylation patterns usually in promoters and enhancers.
  • the target polynucleotide may comprise a mixed polymer of any of the polymers described above.
  • the target polynucleotide may comprise a cyclic RNA DNA polymer of the type used for gene therapy.
  • the length of the target polynucleotide is not important for the function of the invention.
  • the target polynucleotide may thus comprise from 8 to 50,000 bases or even more than 50,000 bases.
  • the choice of length is mainly dependent on the purification and amplification steps, the number of sub-sequences to interrogate with the probes and the distance between such sub-sequences, which may each comprise a polymorphism.
  • usually the target need not exceed the length of the probes with more than 5 nucleotides in the terminal labelled end.
  • the length of the target polynucleotide may thus be selected from 8-15 bases, from 15-30 bases, from 30 to 50 bases, from 50 to 100 bases, from 100 to 200 bases, from 200 to 300 bases, from 300 to 500 bases, from 500 to 750 bases, from 750 to 1000 bases, from 1000 to 1500 bases, from 1500 to 3000 bases, from 3000 to 5000 bases, from 5000 to 10000 bases, from 10000 to 15000 bases, from 15000 to 20000 bases, from 20000 to 25000, from 25000 to 30000 bases, from 30000 to 35000 bases, from 35000 to 40000 bases, from 40000 to 45000 bases, from 45000 to 50000 bases, more than 50000 bases.
  • the whole length of the molecule is intended.
  • the probe only hybridises to one or more relatively short sequence(s) of the target polynucleotides. These sub-sequences to which the probe hybridises are termed the range of overlap between target and probe. This length of the overlap between the probe and target polynucleotide may be as short as at least 5 nucleotides. However more specific hybridisation is obtained by increasing the length of the overlap.
  • the overlap is at least 6 nucleotides, such as at least 7 nucleotides, for example 8 nucleotides, such as at least 9 nucleotides, for example at least 10 nucleotides, such as at least 15 nucleotides, for example at least 20 nucleotides, for example at least 25 nucleotides, such as at least 50 nucleotides, for example at least 100 nucleotides.
  • the overlap is at most 100 nucleotides.
  • Extraction of target nucleic acids can be performed using methods known to those skilled in the art (Joseph Sambrook & David W Russell (2001) "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, USA).
  • extraction protocols among the numerous available protocols, it is preferable to select protocols where the buffer ingredients do not interfere with the recordation of spectral data.
  • Probes used in the method according to the present invention may be made from any kind of nucleotide monomer or any combination of the known types of monomers.
  • the probe may comprise at least one RNA monomer, and/or comprises at least one DNA monomer, and/or at least one PNA monomer, and/or at least one methylated monomer, and/or at least one labelled monomer, and/or at least one LNA monomer.
  • the probe is designed to hybridise specifically to a predetermined sequence in the target polynucleotide if this sequence is present there. Therefore, the probe may be designed to hybridise to any sequence of interest.
  • polymorphisms and/or mutations related to human diseases or animal diseases Any disease or health related problem influenced by genetic factors is envisaged, such as any one or more listed in the International Statistical Classification of Disease and Health Related Problems, ICD-10, of the World Health Organisation. In the following a number of such human diseases with a genetic impact are described.
  • the polymorphism is located at the centre of the sequences.
  • the nucleotide complementary to the polymorphism is located approximately at the centre of the probe as is usually done with probes for detection of polymorphisms.
  • the label is most often bound to either the 3' or 5' terminal nucleotide.
  • the label may also be bound to another nucleotide, e.g. it may be bound to a (non-terminal) nucleotide being complementary to the polymorphism.
  • the nucleotide complementary to the polymorphism When designing probes for use according to the present invention, it is more advantageous to have the nucleotide complementary to the polymorphism positioned terminally than centrally in the probe.
  • the end of the probe When the polymorphism is located terminally, the end of the probe will either be completely complementary to the target nucleic acid sequence and hybridise at all positions, or the nucleotide in the end will be non-complementary and therefore probably bend away from the target nucleic acid sequence.
  • the label is also bound to this terminal nucleic acid, which is either complementary of non-complementary to the target, then the spectral difference in signal between the hybrid with the wild-type and the hybrid with the mutant target nucleic acid sequence is maximised.
  • Another possibility is to design a probe, which does not overlap with the polymorphic site, and which has the label in the position complementary to the polymorphic site. Such a probe will give rise to a spectral difference in signal between wild-type and mutant, since the label will interfere differently with the different nucleic acids in the site.
  • a further possibility is to have the label bound to a non-terminal nucleotide, which is complementary to the polymorphic site.
  • the spectral difference between wild-type and mutant is also enhanced.
  • probes are preferred, where the label is positioned as close as possible to the polymorphic site to maximise the spectral difference.
  • the distance from the label to the polymorphic site may be 1 nucleotide, 2 nucleotides, 3 nucleotides, 5 nucleotides, 10 nucleotides or more.
  • the most preferred are those where the nucleotide complementary to the polymorphic site is in a terminal position.
  • apolipoprotein B mutations related to atherosclerosis wherein the probe may comprise a sequence from any of SEQ ID NO 1 to 4.
  • apolipoprotein E polymorphism (apoE2, E3 and E4) related to neurological diseases, wherein the probe may comprise a sequence from any of SEQ ID NO 5 to 8.
  • the probe may comprise a sequence from any of SEQ ID NO 9 to 10.
  • the probe may comprise a sequence from any of SEQ ID NO 13 to 14.
  • the probe may comprise a sequence from any of SEQ ID NO 11 to 12.
  • Mismatch repair gene mutations related to cancer wherein the probe comprises a sequence from any of SEQ ID NO 15 to 16.
  • the probe may be selective for a mutation in a promoter sequence, or the probe may be selective for a mutation in a coding sequence, including introns and exons.
  • the probes may be used to diagnose the presence/absence and/or nature of a microbial infection, wherein the probe is selective for a microbial target nucleic acid sequence.
  • the probe is selective for a microbial 16S, 18S, or 23S rRNA sequence, because these contain sequences which are conserved across a large group of microbes as well as sequences, which are species-specific.
  • SEQ ID NO 17 is a general probe, which captures 16S RNA.
  • SEQ ID NO 18 is an example of a probe which is specific for all Enterobacteriaceae 16S.
  • SEQ ID NO 19 is a species specific probe which detects E. coli ECA75F. As can be understood from the foregoing, it is possible for the skilled person to design the necessary probes for any desired purpose.
  • the method of the present invention can be used for multiple hybridisation assays in the same vessel, because the contribution to the total spectrum by the various hybrids formed can be resolved by the multivariate analysis. Accordingly, one can use at least two polynucleotide probes capable of hybridising to two different target polynucleotides. Preferably, but not necessarily the two probes are linked to two different detectable labels.
  • the three, four, five or more probes are linked to three, four, five or more different detectable labels.
  • One special embodiment of the multi-probe layout comprises the use of at least one probe having at least two stretches of complementarity to at least one target polynucleotide, such as at least 3 stretches, for example at least 4 stretches, such as at least 5 stretches.
  • the two stretches are separated by a nucleotide sequences, which does not hybridise to the target polynucleotide.
  • Such probes are adapted e.g. for determination of the haplotype of multiple mutations/polymorphisms in the same gene.
  • hybridisation and recording of spectral data is performed in solution. This is because it is not necessary to purify the hybridised polynucleotides and remove e.g. unbound probe. Therefore it is possible to record spectral data from a solution comprising both the hybrid polynucleotide and unhybridised probe. The spectrum form the complex solution is changed by the mere presence of the hybrid.
  • the ratio of target to probe in the hybridisation solution may range from 1 :0.1 to 1 :10, such as 1 :0.2, 1 :0.5, 1 :0.75, 1 :1 ; 1 :2, 1 :4, 1 :5, 1 :7, 1 :8, or 1 :10.
  • the solid support may comprises a solid surface capable of immobilising a capture probe, a capture probe capable of immobilising the target polynucleotide, and a labelled detection probe capable of hybridising to the immobilised target polynucleotide.
  • the capture probe may be is immobilised a priori to the solid surface or the capture probe may be hybridised to the target before immobilisation on a solid support.
  • the capture probe(s) is/are (an) allele specific probe(s).
  • the solid support is a disposable or reusable device such as but not exclusively a flow-through system.
  • a flow through system may comprise immobilised capture probes to capture the target, which can then be labelled by hybridisation with a label probe.
  • immobilised capture probes to capture the target, which can then be labelled by hybridisation with a label probe.
  • the target polynucleotides may be amplified by one of many methods.
  • One of the best known and widely used amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in US 4,683,195, US 4,683,202 and US 4,800,159, however other methods such as LCR ( ⁇ gase Chain Reaction, see Genomics (1989) 4:560-569), NASBA (Nucleic Acid Sequence-Based Amplification, see PCR Methods Appl (1995) 4, S177-S184), strand displacement amplification (Current Opinion in Biotechnology (2001) 12:21-27) or rolling circle amplification (Current Opinion in Biotechnology (2001) 12:21-27) can be applied.
  • Genuine amplification in e.g. bacteria or yeast can also be used to amplify nucleotide sequences as can non-PCR methods, such as T7-polymerase.
  • hybridisation signifies hybridisation under conventional hybridising conditions, preferably under stringent conditions, as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual,
  • stringent when used in conjunction with hybridisation conditions is as defined in the art, i.e. 15-20°C under the melting point T m , cf. Sambrook et al, 1989, pages 11.45-11.49.
  • the conditions are "highly stringent", i.e. 5-10°C under the melting point T m .
  • hybridisation only occurs if the complementarity between a sequence of the polynucleotide probe and a sequence of the target polynucleotide of interest is 100 %, while no hybridisation occur if there is just one mismatch.
  • optimised hybridisation results are reached by adjusting the temperature and/or the ionic strength of the hybridisation buffer as described in the art.
  • LNA locked nucleic acid
  • PNA protein nucleic acid
  • the sugar backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone (Science (1991 ) 254: 1497- 1500).
  • the hybridisation conditions are chosen so that the formation of a hybrid polynucleotide takes place under conditions of optimal or suboptimal stringency providing sufficient stable complexes for discriminatory signal detection, any composition of buffers optimising discriminatory signal detection, any form and concentrations of one or more salts optimising discriminatory signal detection, any additives including but not limited to stabilisers and/or quenchers optimising discriminatory signal detection, temperature range for hybridisation specific for any specific combination of analyte and probe optimising discriminatory signal detection, any range of time of hybridisation necessary to optimise discriminatory signal detection.
  • the hybridisation temperature can be varied within the normal limits.
  • the formation of a hybrid may be performed at a temperature between 10 and 90°C such as 10 to 20 °C, 20-30 °C, 30 to 40 °C, 40 to 50 °C, 50 to 60 °C, 60 to 70 °C, 70 to 80°C, or 80 to 90°C.
  • the buffer used for the hybridisation is conveniently a PCR buffer, which preferably is non-fluorescent, and/or which stabilises the spectrum of electromagnetic radiation, and which allows hybridisation.
  • the methods of the present invention may likewise be used for the determination of haplotypes.
  • the haplotype is the set of alleles borne on one of a pair of homologous chromosomes. Often the particular combination of alleles in a defined region of some chromosome, e.g. the locus of the major histocompatability complex (MHC), is referred to as the haplotype of that locus.
  • MHC major histocompatability complex
  • the central dogma of modern molecular genetics teaches that it is the haplotype of the coding part of a gene that determines the amino acid sequence and thus the function of the resulting protein.
  • haplotype-investigations are superior to conventional genetic investigations of only single loci, because the haplotype provides physiological relevant information from more loci.
  • the determination of the haplotype may for example be carried out using allele specific primers to amplify only one of two alleles. This is done by using a primer set, wherein at least one of the primers is 100% complementary to only one allele and carrying out the annealing step of the amplification at high stringency, so that hybridisation will only take place between the primer and the allele being 100 % complementary to the primer.
  • the presence of a further polymorphism in the amplified fragment can be determined using an allele specific polynucleotide probe.
  • both alleles are isolated and amplified (if required) and the two strands are separated using allele specific capture probes.
  • the presence of a further polymorphism on the separated fragments can be determined using an allele specific polynucleotide probe.
  • both the resolution of the haplotypes and the detection of the multiplicity of nucleotide polymorphisms are performed in suspension, defined here as a "one-phase system". In one embodiment this is accomplished by the amplification of the preselected region.
  • the amplified fragment is hybridised to specially designed probes which are capable of detecting a multiplicity of polymorphisms.
  • Such "bifunctional" probes contain at least 2 stretches of relatively short sequences which are complementary to each of the two studied polymorphisms separated by a region of spacer DNA which will not hybridise with the amplified region under the conditions in the experiment.
  • the procedure takes advantage of the fact that intramolecular hybridisation is thermodynamically favorable compared to hybridisation between separate molecules.
  • This difference between intra- and inter-molecular hybridisation is in particular significant when hybridisation is performed at low concentrations of nucleic acid and results in a significant difference even between the hybridisation of the bifunctional probe to one or two amplified fragments, the hybridisation to one fragment being the most favourable.
  • Under stringent conditions employed in the hybridisation only probes which are completely complementary to the sequences comprising both studied polymorphisms will form stable hybrids.
  • the haplotype can be determined by recording a fluorescence spectrum from the different oligonucleotides hybridised to the amplified DNA without the need for separation of hybrid from .
  • the invention also relates to a kit for detection of mutations or polymorphism comprising at least one oligonucleotide probe capable of hybridising to a preselected region of a target polynucleotide, the polynucleotide probe further comprising at least one detectable label, instructions enabling correlation of spectral data recorded from a hybrid polynucleotide between said at least one oligonucleotide probe and said target polynucleotide to the presence or absence of said mutation or polymorphism using multivariate analysis.
  • the kit is assembled and sold with probes and instructions enabling the performance of one of a number of different assays as outlined above.
  • the instructions can be regarded as calibration data, which enable the user to perform the hybridisation assay without first determining the difference between a spectrum from an unbound an a bound probe.
  • the instructions are generally rather complex and may conveniently be stored on a data storage medium, which is provided together with the probes, and optional buffers. Instructions may be in the form of calibration data on a data carrier, such as floppy disc, a CD-ROM, a DVD, ROM, chips, memory-cards, bar-codes.
  • kits may then comprise the address of a propagated signal comprising calibration data, which can be transferred over a network, such as e-mail, internet, on-line nets, fibre-optics, power-cables, satellite- dishes.
  • a network such as e-mail, internet, on-line nets, fibre-optics, power-cables, satellite- dishes.
  • the oligonucleotide probes comprised in the kit may also in one embodiment be a multifunctional probe having two or more sequences which can hybridise to separate sequences in the target. The two or more sequences are separated by a spacer sequence, which does not hybridise to the target. Examples of such multifunctional probes can be found in PCT/DK02/00552 (Hvidovre Hospital).
  • kits may also comprise two or more differentially labelled probes, which hybridise to different sequences in the target nucleotide. Such differently labelled probes can be used for determining the linkage phase of two or more mutations/polymorphisms.
  • kits will additionally comprise at least one control polynucleotide capable of hybridising to the oligonucleotide probe and non-hybridising polynucleotide(s).
  • the probes in the kits can be in the form of probes, which can be used in solution or the probes may be linked in one end to a moiety, which may be used for immobilising the probes to a solid surface, as described above.
  • the kit may also be in the form of a tube with at least one probe linked to the inner surface, the tube wall allowing electromagnetic radiation to pass the walls.
  • the tube is made in a shape which fits into a standard spectrophotometer.
  • Such a tube may comprises more than one probe linked to more than one location, the locations being spatially separate.
  • the tube may comprise a probe selective for a wildtype in one location and a mutation/polymorphism in another location.
  • the tube may also comprise more than one probe the probes having detectably different labels, which preferably do not interfere with each other's spectrum. In this way hybridisation to the two probes can be detected with one scan over a given wavelength range.
  • the invention in a further aspect relates to a system for detection of a hybrid polynucleotide comprising at least one oligonucleotide probe being at least partly complementary to a target polynucleotide, the probe comprising a detectable label, a sample chamber from which electromagnetic radiation can be recorded, a source of spectrally resolved electromagnetic radiation, means for sensing and recording a spectrum of electromagnetic radiation from the sample chamber, a computer unit for storing spectral data of electromagnetic radiation and having instructions to treat the recorded spectral data using multivariate analysis.
  • the system may further comprising a computer controlled robot to transfer solutions to the sample chamber.
  • the system comprises means to control the temperature of the sample chamber during hybridisation and subsequent recording of spectrum.
  • the sample chamber may be in the form of a tube with at least one probe linked to the inner surface.
  • the tube may comprise more than one probe linked to more than one spatially separate location and the system comprises means to record a spectrum from each of the spatially separate locations.
  • the tube may comprise more than one probe, the more than one probe having detectably different labels.
  • the system preferably is made to accommodate standard laboratory glass and/or plasticware, and may in one preferred embodiment be adapted to accommodate a multi-well dish and record a spectrum for each well.
  • the multi-well dish may be a 96 well dish a 384 well or a standard dish with more wells.
  • a solid support may also comprise a dish or a rod, spinning dishes or rotating and displaceable rods.
  • Capture-probes are attached to the dish or the rod in predefined areas so that the hybridised samples are unequivocally identified. Only one or a few spectral detection-units are needed as the spinning of the dish or rod will bring all the samples into focus of the detection device.
  • the oligonucleotides used for the examples were selected from a region of the Apolipoprotein gene - Apo3611.
  • the oligos are aligned in Figure 4 to show the regions of complementarity.
  • Wildtype 2 (SEQ ID NO: 21) 2 5 'ACCAGAAGATCAGATGGAAAAATGAAGTCCGGATTCATTCTGGGTCTTTC
  • Non-specific target molecule SEQ ID NO: 33
  • Rhodamine labelled oligonucleotides Rho- 5 ' -CGGACTTCATTTTTC ( SEQ ID NO : 38 )
  • Rho- 5 ' -TGGACTTCATTTTTC SEQ ID NO : 39
  • Rho-5'-ATGAATCTGGACTTC SEQ ID NO: 41
  • DNA target and fluorophore labelled oligonucleotides (usually 0.5 ⁇ M) were kept at the hybridisation temperature in a commercially available hybridisation buffer containing 1 X PCR buffer - 10 mM Tris-HCI pH 8.3, 50 mM KCI (Perkin Elmer, catalog # N808-0010).
  • Spectrofluorometry Equal volumes of target DNA and fluorophore labelled oligonucleotide were mixed in a tube and kept at the hybridisation temperature for 5 min until reading of the spectrum. The final concentrations of all oligonucleotides were 0.25 ⁇ M, unless otherwise stated. Spectra were obtained using a Perkin Elmer LS50 Spectrofluorometer. The cuvette holder and the plastic cuvette (VWR (Merck) catalog # 122455/100) was kept at a constant temperature (23°C, 30°C or 45°C) by connection to a termostated water bath. Individual oligonucleotide mixtures were kept at the hybridisation temperatures in a termostated water bath. Spectral ranges are provided in Table 5. The excitation spectrum was obtained by keeping the Em max constant, whereas the emission spectrum was obtained by keeping the excitation wavelength at Ex max .
  • a Cy5 labelled oligonucleotide (complementary to the wildtype target) was hybridised to the wildtype target (W2), the mutant target (M) or the dummy target (Z), respectively. Spectra were obtained at 30 °C after 5 min of hybridisation.
  • Cy5 labelled oligonucleotides with LNA monomers are hybridised to a polymorphic site in the muscle glycogen synthase promoter.
  • Table 6 summarises the oligonucleotide compositions used in each of the of 5 experiments.
  • the nomenclature of the oligonucleotides is deciphered above. Terminally implies that the mutation is addressed by the nucleotide, which is placed at the 5'-end attached to the fluorophore via a linker. Internally means that the mutation is addressed by an unlabelled nucleotide placed in the centre position of the oligonucleotide.
  • Figure 4 gives an overview of the target wildtype and mutant DNA and of all (partially) complementary labelled oligonucleotides used in the present examples.
  • Figure 5 shows, that when the 4 different oligonucleotides (A, B, C and D) are hybridised to a target DNA (W1 ) it changes their placement in the scores plot - i.e. the hybridisation changes the fluorescence spectra.
  • the first prefix (1 or 2) denotes the replicate number
  • the second prefix only represent two fluorescence measurements on that same replicate solution. This implies, that samples with the prefix 1.1 and 1.2 and the prefix 2.1 and 2.2 respectively should be placed closer to each other in the plot - only showing the difference in fluorescence spectra obtained successively in time. The plot shows that this is the case. There is most often a larger variation within replicates than within two fluorescence measurements on the same replicate. In conclusion there are some differences between replicates and double measurements. It should be noted that the hybridisations reactions were not allowed to hybridise for exactly the same time in this first shot experiment. Conclusion of experiment 1 :
  • Hybridisation changes the fluorescence emission spectra.
  • the 4 hybridised oligonucleotide combinations results in different emission fluorescence spectra.
  • Figure 6 shows, that when the 4 different oligonucleotides (A, B, C and D) are hybridised to a target DNA (W1) it changes their placement in the scores plot - i.e. the hybridisation changes the obtained fluorescence spectrum (emission).
  • a Cy5 labelled oligonucleotide (complementary to the wildtype target) was hybridised to the wildtype target (W2), the mutant target (M) or the dummy target (Z), respectively. Spectra were obtained at 30 °C after 5 min of hybridisation.
  • Figure 10 shows, that when oligonucleotide A are hybridised to a target DNA (W2 or M) it changes its placement in the scores plot - i.e. the hybridisation changes the obtained fluorescence spectrum (excitation). Note that the non-specific target (Z) groups near to A alone.
  • the 3 hybridised oligonucleotides (A-W2, A-M and A-Z) is placed in each group in the scores plot - i.e. the 3 hybridised oligonucleotides may represent different fluorescence spectra.
  • Hybridisation changes the fluorescence spectrum, (both the excitation and the emission spectra).
  • the 3 hybridised oligonucleotides (A-W2, A-M and A-Z) separates out in different groups.
  • the 2 hybridised oligonucleotides A-W2 and A-M are placed in each group in the scores plot.
  • the hybridised oligonucleotides A-W2-M (W2 and M targets are present simultaneously) are placed in between the A-W2 group and the A-M group.
  • No clear difference between the two W2-M concentrations was observed, i.e. the two concentrations (0,25 or 0,125 ⁇ M of each oligo nucleotide) did not influence on the grouping.
  • Figure 13 (emission spectrum) gives the same picture as is depicted in Figure 12. Compared to the results observed in Figure 12 which is based on excitation spectra, the results based on the emission spectra indicates a smaller variation within each group in relation to the variation between the groups.
  • Hybridisation changes the fluorescence spectrum, (both in excitation and in emission).
  • A-W3 and F-A-W3 lie within the same group i.e. the Cy5 spectrum is relatively unaffected by the presence of the Cy3 labelled oligonucleotide. This is supported by the observation that A and A-F lie within the same group. Note that A-F is distinct from A-F-W3 as opposed to the situation on the excitation spectrum (Fig. 14) where they were grouped.
  • Hybridisation changes the fluorescence spectrum, (both in emission and in excitation).
  • F-A and F-A-W3 are grouping close together
  • the technique is stable over time, changes of reagents and independent of specific technical skills of the staff.
  • the major impact of letting the same person perform the data analysis was to reduce the time used to a few minutes.
  • dsDNA was created by hybridising equimolar amounts of oligonucleotide W2 to its complementary strand R ( Figure 4) mixing 1 ⁇ M solutions of the two strands in standard buffer and incubated for 10 minutes at 30°C.
  • the dsDNA was denatured by heating to 94 °C for 10 min and cooled on ice. Equimolar amounts of Cy5- labelled probe A was added, and the reaction mixture was incubated for 5 minutes at 30°C The spectra and data analysis was performed as previously described.
  • the hybridisation mixture groups the wildtype dsDNA-probe complex (AW2-R) and the mutant dsDNA-probe complex (AM-Rm) in two clearly separated areas of the score plot.
  • AW2-R- denotes dsDNA not denatured before the reaction with the probe.
  • Hybridisation to single stranded DNA (AM and AW2) and non-specific single stranded DNA (AZ) is included for illustrative pur- poses. These latter complexes are not normally present in the reaction vial. The most consistent grouping is seen with dsDNA targets denatured before annealing to the labelled probe.
  • Hybridisations with dsDNA as target changes the fluorescence spectra.
  • the wildtype and mutant dsDNA complexes grouped in separate groups, suggesting that the Cy5-labelled probe displaced the complementary strand, i.e. no elimination of the complementary strand was necessary. This means that the reaction can be per- formed as a homogenous reaction with no purification steps needed.
  • WA Poly2 antisense wildtype (SEQ ID NO: 61): ⁇ '-atggggccagacccgagattctgggatcccagccccCtccccgcctcagatccagaagtccagc
  • WS Poly2 sense wildtype (SEQ ID NO: 62): 3' -taccccggtctgggctctaagaccctagggtcgggggaggggcggagtctaggtcttcaggtcg
  • MA Poly2 antisense mutant (SEQ ID NO: 63): 5' -atggggccagacccgagattctgggatcccagccccGtccccgcctcagatccagaagtccagc
  • WP Poly2 wild type probe (SEQ ID NO: 65): Cy5-5'-Gggggctgggat
  • dsDNA was created as described in above. Reaction conditions was exactly as described in the previous experiments.
  • Figure 20 shows the score plot (excitation) from the reaction mixtures. The complex between the probe and the target was grouped in separate areas for the wild type and the mutant.
  • PCR amplification reaction
  • the amplification reaction (PCR) in 25 ⁇ L consists of 100 ng of target, 1 ⁇ M of primer 3611 s and 3611 as each, 0.5 unit Taq polymerase (Quiagen Core), 0.2 mM of each of the four dNTP, 1.5 mM MgCI 2 , 5 ⁇ l Q-solution (from Quiagen), and PCR-buffer accompanying the Taq polymerase.
  • the PCR-reaction is performed with an initial denaturation at 94°C for 5 minutes, 35 cycles of denaturation at 94°C for 40 seconds, annealing at 56°C for 40 seconds and extension at 72°C for 40 seconds.
  • the final extension step is extended to 10 minutes.
  • the reaction solution is heated to 94°C for 5 min- utes, cooled on ice, and the Cy5 labelled probe A is added to a final concentration of
  • the solution is diluted by PCR-buffer to 500 ⁇ L and incubated at 30°C for 5 minutes and the spectra was recorded and data processed as previously.
  • Highlighted residues correspond to the position of polymorphisms in the target nucleic acid sequence.
  • Apolipoprotein B 3500 mutation SEQ ID NO 1 5'-AGCACACGGTCTTC (wt)
  • Apolipoprotein B 2488 polymorphism is Apolipoprotein B 2488 polymorphism
  • Apolipoprotein E polymorphism (apoE2, E3 and E4) related to neurological diseases
  • Dnaset mutations related to rheumatological diseases SEQ ID NO 11 5'-GGGGCATGAAGCTGCT (wt)
  • SEQ ID NO 12 5'-GGGGCATGTAGCTGCT (mutation)
  • methylene tetrahydorfolate reductase polymorphisms related to osteoporosis SEQ ID NO 13 5'-TGCGGATCGATTTC (wt)
  • SEQ ID NO 17 5'-AGGAGGTGATCCAACCGCA (general capture-probe for 16S)
  • SEQ ID NO 18 5'-GGCGCTTACCACTTTGTGATTCAT (specific capture-probe for Enterobacteriacea 16S)
  • SEQ ID NO 19 5'-GGAAGAAGCTTGCTTCTTTGCTGAC (specific capture-probe for E. Coli-ECA75F 16S)
  • SEQ ID NO 20 W1 5-ctaagaaccagaagatcagatggaaaatgaagtccggattcattctgggtctttccagagccaggtcga
  • Apolipoprotein E polymorphism (apoE2, E3 and E4) related to neurological diseases (Jarvik 357-62)
  • the residue corresponding to the polymorphism is located in the 3' end of the probe.
  • the label may be linked to the 3' or the 5' terminal residue.
  • SEQ ID NO 42 5'-ATGGAGGACGTGT apoE codonl 12-cys
  • SEQ ID NO 43 5'-ATGGAGGACGTGC apoE codonl 12-arg
  • SEQ ID NO 44 5'-GACCTGCAGAAGT apoE codon 158-cys
  • the label is located at the position of the polymorphism.
  • SEQ ID NO 48 5'-CCAGGCGGCCGCA apoE codonl 12-cys
  • SEQ ID NO 49 5'-CCAGGCGGCCGCG apoE codonl 12-arg
  • SEQ ID NO 50 5'-ACACTGCCAGGCA apoE codonl 58-cys
  • SEQ ID NO 51 5'-ACACTGCCAGGCG apoE codonl 58-arg
  • Probes with the label in the position of the polymorphism Hybridise to the opposite string as compared to Y1 -Y2.
  • SEQ ID NO 53 5'-TACACTGCCAGGC-label apoE codonl 58-cys/arg Probes with the residue complementary to the polymorphism in a central position. The label may be in the 3' or 5' end. These probes hybridise to the opposite string compared to SEQ ID NO 5 to 8.
  • SEQ ID NO 54 5'- GCGGCCGCACACGTC apoE codonl 12-cys
  • SEQ ID NO 55 5'- GCGGCCGCGCACGTC apoE codonl 12-arg
  • SEQ ID NO 56 5'- TGCCAGGCACTTCTG apoE codon 158-cys
  • SEQ ID NO 57 5'- TGCCAGGCGCTTCTG apoE codon 158-arg

Abstract

L'invention concerne des procédés, des trousses et des systèmes permettant de déterminer la présence ou l'absence de polynucléotides cibles par hybridation avec des sondes polynucléotidiques comprenant une étiquette détectable suivie d'une analyse spectrale, de préférence une analyse multivariable. L'analyse spectrale permet de déterminer si la sonde fait ou non partie d'un polynucléotide hybride et, par conséquent, si le polynucléotide cible est présent ou non. De plus, l'analyse des données spectrales permet de déterminer, dans le cas où la sonde appartient à un polynucléotide hybride, s'il existe ou non un ou plusieurs mésappariements entre la sonde et la cible. Ces procédés, trousses et systèmes peuvent servir à déterminer des mutations et des polymorphismes.
PCT/DK2003/000594 2002-09-13 2003-09-12 Procede de detection rapide de mutations et de polymorphismes nucleotidiques par chimiometrie WO2004024949A2 (fr)

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