Patent Application
20050164407
DNA hybridization is based on the sequence-specific formation of a complementary double strand from different single-strand sources under particular experimental conditions. If a single strand sequence is known and utilized as a probe (for example in the form of an oligonucleotide), it is possible, after detection of a hybridization event, for example via labeling with a dye, to derive the target sequence. This process is reversible and may be controlled via changes in temperature. This property of DNA is utilized in various applications, for example in DNA sequence decoding (Sequencing by hybridization, SbH) or in measuring the activity of different cells or of different cellular states (Expression Profiling or Gene Expression Monitoring), by determining the copy number of DNA transcripts (mRNA) which are present in a cell at a defined time. In this connection, hybridization events of single-stranded DNA and of mRNA need to be evaluated quantitatively. Another important application, a special case of sequencing, is the mutational analysis of individual DNA single positions (Single Nuclear Polymorphisms, SNP) which serve to elucidate diseases at a molecular level.
For practical applications, the so-called “nucleic acid chip technique” has been established as a promising tool for the abovementioned problems. This involves immobilizing hybridization probes of a known sequence, for example oligonucleotides or cDNA molecules, at defined locations on surfaces of a support, which probes are used there as capture molecules for target sequences from different sample material. Via detection of the hybridization of target sequences to said surfaces, for example by labeling the sample material with fluorescent dyes, a hybridization experiment produces measured signals which may be evaluated with the aid of suitable methods. Owing to the fact that the probe sequence is known, it is possible to identify and characterize the target sequences in the sample material.
Hybridization to a solid phase, for example a DNA chip, DNA array or DNA filter, is a diffusion-dependent process which depends on a complex combined action of various factors, inter alia
With practical application, for example in expression profiling, SbH and others, owing to the complexity of the DNA molecules in the sample material, to the usually large sample volumes and chip areas, strong limitations are encountered with respect to:
Nucleic acid hybridization is an equilibrium process which may be described by the law of mass action: [A] (probe)+[B] (target sequence)⇄[AB]. Since [A], i.e. the concentration of the probe immobilized on a chip, is, according to the prior art, usually approximately constant for all immobilized probes (A1 to An), problems arise for the relative quantification of target sequences (B) in sample mixtures (B1 to Bn), if [B1] to [Bn] (i.e. the concentration of the individual target sequences B1 to Bn) is not constant. This is the case, for example, in gene expression profiling. Individual target sequence concentrations may vary by a factor of 10 000. As a result of this, some probe locations on the chip may be physically saturated in an experiment, in comparison with other probe locations, and the linear dynamic measuring range is exceeded during detection, thereby rendering impossible a quantification of all signals in a single measurement. This has an influence on the sensitivity (quality) and possible applications (utilization).
In the SbH application, for example, there is the problem that, in the case of eukaryotic target sequences, a large amount of repetitive sequences (97% in the human genome) is present, as a result of which the relative concentration of the relevant target sequence region is almost by a factor of 100 lower than it could be, if, for example, the repetitive sequences were to be “filtered out” prior to the experiment. This has an influence on the sensitivity and specificity (quality) and impedes many conceivable applications using DNA chips.
Another problem of the SbH application is the fact that it is not possible in principle to specifically form any desired DNA double strands at a defined hybridization temperature (quality, utilization), since DNA hybridization is kinetically controlled and double strands form which do not correspond to the thermodynamic minimum. Only by reversibly dissolving unspecifically bound DNA molecules and by setting the individual duplex melting temperature, may the reaction in the direction of the thermodynamically most favorable state be made possible (specific double strand formation).
Studies on the determination of analytes on solid phases are known, as described, for example, by R. P. Ekins, in U.S. Pat. No. 5,432,099. The known DNA chip hybridization methods may be divided into two categories: the passive hybridization method and the actively supported hybridization method.
In passive hybridization, the sample solution is stationary and the hybridization is carried out at a defined temperature in a diffusion-dependent manner. This category includes the two-dimensional slide or array technique using chips which are prepared by spotting or in situ synthesis. These techniques have the advantage of having a relatively high location density. Disadvantageously, however, the use of large sample volumes is required, only a low local target sequence concentration causing, inter alia, a very slow hybridization (approx. 16-48 h) is produced and the usable linear measuring range covers only 2-3 orders of magnitude. Another disadvantage arises due to the two-dimensional uniform temperature which may result in unspecific (false-positive) hybridization results. In the case of SbH with repetitive DNA, the signal-to-background ratio ranges from low to not measurable in this technique.
In the case of the actively supported hybridization methods, the sample solution is moved through channels or, with the aid of electric fields, across the immobilized probes, and a temperature gradient can be set. This category includes the 3D chip technique with channel geometry. This technique is advantageous in that the hybridization times are short, due to the active movement of the sample, and that relatively small sample volumes can be utilized. Disadvantageously, however, the location density is low and a local temperature control cannot be set, which may result in false-positive events.
Another actively supported hybridization method is the “96-well printing” technique in microtiter plates. This technique has the advantage of the individual microtiter plate wells being individually thermally controllable, for example with the aid of a PCR apparatus. However, disadvantages are the use of very large sample volumes, the low location density and the diffusion-dependent and slow hybridization which strongly affects the sensitivity of this method.
Finally, the electronically controlled hybridization is known. This type of actively supported hybridization involves moving the sample toward the probe. As a result, the hybridization times obtained are very short. Due to the electronics, a temperature-equivalent stringency is produced, making a discrimination of false-positives possible. Nevertheless, this technique is quite expensive, has very low integration densities and cannot readily be used for SbH and genomic expression profiling.
In summary, it can be pointed out that previously established methods of detecting nucleic acids by hybridization using chips with immobilized hybridization probes cannot be adequately adapted to the complexity of biological sample material. It was therefore an object of the present invention to provide methods and systems for the determination of analytes on supports, for example chips, which methods and systems at least partially avoid the disadvantages of the prior art.
This object is achieved by a method in which individual regions or groups of regions of hybridization probes on the support are designed in a variable manner for the application desired in each case, thus considerably improving the sensitivity, specificity and economy. The method of the invention, however, enables not only the analytes to bind to a probe by hybridization but also other receptor-analyte bioaffinity interactions such as, for example, nucleic acid-protein, protein-protein, low molecular weight compound-protein or receptor-ligand bonds to be detected.
Thus, the object of the present invention is a method of determining analytes, comprising the steps
Preferably, the method of the invention is based on the Geniom® technology which is described in WO 00/13018. It may utilize the geometric structures (micro-channels), the flexible loading capacity of the fluid processor (i.e. the different local receptor concentrations as depicted in
Especially the production times of new DNA chips, which are particularly short with the Geniom® technology (within a few hours), and the short learning cycles made possible thereby render these applications with DNA chips not only technically feasible but also economical, such as, for example, sequencing (SbH) of DNA with a high proportion of repetitive DNA, expression profiling with a sufficient dynamic sensitivity range, in order to be able to record quantitatively both very rare and very common transcripts in complex mRNA samples, and massively parallel SNP detection with high individual oligo duplex specificity.
It is possible in the method of the invention, preferably by using the Geniom® technology, for example using an integrated synthesis and analysis system (ISA system), to vary biophysical parameters such as temperature and local and virtual concentration—alone or in combination—both in the preparation of a test and during a test (online detection) or/and in the evaluation of a test (learning system). This manipulation of the parameters influencing the hybridization signal may be carried out both globally and locally (i.e. individually for each oligonucleotide sensor).
Anmother object of the invention is an apparatus for determining an analyte, comprising a support having a plurality of predetermined regions at which in each case different receptors are immobilized on said support, characterized in that said predetermined regions with receptors have, at least partially, a different local receptor concentration. The apparatus of the invention is furthermore characterized in that means are provided in order to vary the kinetics of the receptor-analyte interaction or/and to vary the virtual analyte concentration in the predetermined regions.
The method of the invention may achieve an improvement, for example in the expression profiling application. Constitutively highly expressed gene sequences may be depleted over areas which are up to 100× larger than the others and have up to 10× higher location densities. This increases the sensitivity for rarely expressed genes. This sensitivity may be optimized by learning cycles, with a new chip being programmed for the genes identified in a first experiment, on the basis of relative frequency (fluorescence intensity), which chip evens out differences via the size or/and receptor density of the locations so as to produce a preferably homogeneous measured signal. The sensitivity may also be optimized by means of different times (amounts) of illumination per location, using a light source matrix. This illumination setting may then be used for studying test material. The system becomes more sensitive and the dynamic range is shifted into the linear range.
An improvement may also be achieved in the SbH application. Repetitive DNA may be depleted in regions of the chip by immobilizing, on suitably large surfaces, special spacers which have relatively high 3-dimensional branching and a relatively high local location density and thus “filter out” the repetitive sequences. This renders the actual measurement more sensitive and delivers a better signal-to-background ratio. This effect may be accelerated by very fast reassociation kinetics: a hybridization is carried out within one minute so that frequently occurring sequences can quickly find their probe. The solution is subsequently removed and stored intermediately in a reservoir (see
When a plurality of overlapping or nonoverlapping receptors for a gene are arranged in proximity of one another, it is possible, via differently sized surfaces of individual receptors, to utilize the effect of mass action in order to balance differences in affinity, i.e. the local concentration of an oligonucleotide probe is varied, meaning that the differences in the melting temperature of various oligonucleotides are compensated for, for example by individually adapting the location size. Thus, a correspondingly larger location area is assigned to an oligonucleotide probe with lower melting temperature, caused, for example, by a high AT content, than to a probe with a higher melting point, caused for example, by a higher GC content. During a subsequent signal quantification, the larger location areas may be integrated and assessed like a standard signal (learning principle).
Different melting points of receptor probes may also be adjusted by varying the area density, in addition to altering the area. This is accomplished, for example, by setting the local receptor density via branched (dendrimeric) structures (cf.
In a first embodiment of the method, one or more predetermined regions are designed with receptors in a different way, i.e. different conditions are chosen for the local receptor concentration from different region sizes, i.e. location sizes for individual receptors, or/and different receptor densities within said regions. According to the invention, those regions which occur frequently in the sample for binding of molecules, for example regions which serve to bind repetitive sequences or regions which serve to bind constitutively highly expressed genes, have an increased local receptor concentration.
Different location sizes may be implemented by way of differently sized synthesis fields during synthesis of the receptors, for example by using an appropriate software. Preferably, the sizes of the individual regions are varied by at least 50%, particularly preferably by at least 100% (based on the size of the smallest region) (see, for example,
Different location densities may be implemented via synthetic chemistry using different reagents, for example spacers with different degrees of branching (see, for example,
Previous methods do not make possible any large variation possibilities regarding the amounts or local concentrations of the probes, so that it is not possible to carry out an individual adaptation to the greatly varying sample material. The variability described herein of the location area and even of the local receptor concentration per location(location density), which variability may be as large as desired, enables, with the aid of two learning cycles, an adaptation to a defined sample material in order to optimize measurement sensitivity.
Furthermore, individual regions or groups of regions with receptors may have different conditions for receptor-ligand affinity. This is implemented by different receptor lengths or/and different types of receptor building blocks, for example PNA or LNA building blocks, in the individual regions. The receptor length of individual regions is preferably varied by at least 20%, particularly preferably by at least 50% (based on the region having the shortest receptor length).
In a further embodiment, different conditions for the kinetics of receptor-analyte interaction are set in one or more predefined regions with receptors, for example selected from different temperatures or/and temperature profiles in said regions or/and different fluid conditions in said regions.
The temperature may be varied across the entire support, for example across the entire area as a stationary or fluctuating temperature gradient or/and locally across individual regions or groups of regions, for example position-specifically. The control of the temperature over the entire area may be implemented with the aid of a Peltier element or by means of thermally controlled air flow. The temperature may be controlled locally by location-specific irradiation of energy, for example as IR radiation with the aid of a light source matrix, involving illuminating individual locations with an individually set amount of light, resulting in heat production due to absorption. The irradiation here is proportional to the formation of heat and increase in temperature. Alternatively or additionally, the local location area temperature may be regulated by electron flows in conductor tracks which run in the support across individual regions. According to the invention, this temperature control also enables a fluctuating temperature gradient to be set in the individual regions or groups of regions with receptors.
Different double strands which are produced by hybridization of receptors to the supports and target sequences in the sample, which sequences are to be analyzed in a parallel process on a single chip, have different hybridization kinetics and melting curves. The fluctuating temperature gradients described herein and temperatures which can be set locally and individually, for example with the aid of a light source matrix, solve this previously “fundamental” problem of specificity in parallel measurements.
According to the invention, the temperatures in the individual regions are varied preferably by at least 2° C., frequently by at least 5° C. and, in some cases, by at least 10° C.
Another possibility of varying the conditions for the kinetics of receptor-analyte interaction in individual regions of the support is the setting of different fluid movements in one or more different regions of the support. This may involve actively moving the sample during the hybridization process in the fluid processor, for example with the aid of pumps (piston pumps, gas pressure pumps). Preferably the sample is actively moved across the support in a circular flow or/and in a rocking movement.
In the method of the invention, the fluid velocity in individual regions of the support is preferably varied by at least 20%, preferably by at least 50% (based on the region having the lowest fluid velocity).
Active fluid movement enables the sample to be actively moved passed the probe, thereby firstly increasing the rate of hybridization and secondly enabling a separation principle to be utilized in order to separate differently hybridizing sample elements from one another after hybridization (chromatographic principle). In this way it is possible, in combination with fluctuating temperature gradients, to increase specificity and sensitivity. Consequently, according to the invention, the sample may be recycled once or several times across the support under various kinetic conditions. In this context, an increasing temperature profile or/and a decreasing temperature profile or/and a combination of increasing and decreasing temperature profiles may be set per cycle.
Another parameter which may be varied in the method of the invention is the virtual analyte concentration. To this end, different conditions for determining the analyte concentration are generated. These comprise generating or/and detecting the measured signal in individual regions with different intensity. Preferably, the analyte is detected by way of fluorescence and the different intensity of the measured signal is generated by locally different irradiation with excitation light, preferably via a light source matrix. According to the invention, the individual illumination intensity of the regions varies preferably by at least 50%, particularly preferably by at least 100% (based on the region having the lowest illumination intensity). The locally variable illumination according to the invention via a light source matrix is diagrammatically depicted in
Previous methods do not enable any individual illumination of individual locations to be controlled to adapt the fluorescence intensities via the amount of excitation light (different illumination of individual locations). After a first test measurement, individual locations may be individually illuminated with the aid of the light source matrix, making it possible to balance different fluorescence emission intensities in individual regions so as not to exceed the linear dynamic measuring range of the detector, for example a CCD camera. This results in an increase especially in the sensitivity and accuracy of quantitative measurements. For example, locations with receptors which have lower melting temperatures are illuminated for a longer time and those with a higher melting point are illuminated for a shorter time so that the signals of the two probes have a comparable intensity. This is particularly important also for applications which do not require quantitative evaluation but are based on a yes/no decision. Examples of these are SNP analyses or resequencing applications in which particular target sequences have a problem, i.e. they are difficult to access for hybridizations, respectively, the corresponding hybridization signals are small and are thus required to be enhanced, and this may then be carried out using local longer illumination times.
According to the method of the invention, the support is preferably a flow cell and/or a microflow cell, i.e. a microfluidic support with channels, preferably with closed channels, in which the predetermined locations with the in each case different immobilized receptors are located. The channels preferably have a diameter in the range from 10 to 10 000 μm, particularly preferably from 50 to 250 μm, and may be designed in principle in any form, for example with a circular, oval, square or rectangular cross section.
The receptors are preferably selected from biopolymers such as, for example, nucleic acids such as DNA and RNA or nucleic acid analogs such as peptide nucleic acids (PNA) and locked nucleic acids (LNA) and also from proteins, peptides and carbohydrates. Particular preference is given to selecting the receptors from nucleic acids and nucleic acid analogs, with binding of the analytes comprising a hybridization.
The method of the invention comprises parallel determination of a plurality of analytes, i.e. a support is provided which contains a plurality of different receptors which may react with in each case different analytes in a single sample. Preference is given to determining by the method of the invention at least 50, preferably at least 100, analytes in the sample in parallel.
The method of the invention is advantageously carried out using an apparatus comprising:
An apparatus of this kind is a light emission detection device disclosed in the German patent applications 198 39 254.0, 199 07 080.6 and 199 40 799.5, which is combined into one apparatus so as to carry out therewith the method of the invention in the form of a cyclic integrated synthesis and analysis. Particular preference is given to using in the apparatus of the invention a programmable light source matrix selected from a light valve matrix, a mirror array and a UV laser array. It is possible to use in the apparatus of the invention two light source matrices, one serving to control the temperature and the other one to detect the measured signals, in the case that the analyte is detected by way of fluorescence. Further preference is given, according to the invention, to using a programmable detection matrix selected from a CCD array, light-sensitive semiconductor structures and electronic detectors. The apparatus of the invention may be utilized for controlled in-situ synthesis of the receptors. Synthesis of the receptors comprises conducting fluid containing receptor synthesis building blocks across the support, location- or/and time-specifically immobilizing said building blocks at the in each case predetermined regions on said support and repeating these steps, until the desired receptors have been synthesized at their in each case predetermined regions. Receptor synthesis furthermore comprises at least one fluid-chemical reaction step or/and at least one illumination step or/and an electrochemical reaction step or/and a combination of such steps.
The present invention will furthermore be illustrated by the following figures:
Supports are prepared, containing in each case 500 locations for GAPDH, actin, and other genes known to be highly expressed and having in each case one location for all other, rarely expressed genes. A hybridization experiment is carried out. According to the measured signal intensities, the individual locations and the individual location densities are adjusted (equilibration of melting temperatures). The hybridization process is repeated, prolonging the detection times in order to increase the sensitivity in the linear measuring range. The redundant locations are integrated to give one measured value.
Half of a support is charged with a multifunctional spacer and with receptor mixtures which are complementary to the repetitive regions and to the vector sequence. These receptors have a length of up to 50 bases. The other half of the support is charged with shorter receptors in order to resequence regions from target genes. A temperature gradient is applied, with elevated temperatures being set for repetitive regions, i.e. for long receptors, in order for these regions not to be depleted of specific sequences due to false hybridization, and low temperatures being set for specific regions, i.e. short receptors. The BAC DNA is randomly fragmented and the hybridization process is carried out subsequently. The hybridization may be carried out cyclically in order to make use of the effect of reassociation kinetics. The signals are detected in the specific range with an improved signal-to-background ratio.
This example is diagrammatically depicted in
This example is depicted diagrammatically in
This example is depicted diagrammatically in
| Number | Date | Country | Kind |
|---|---|---|---|
| 102087709 | Feb 2002 | DE | national |
The invention relates to a method of increasing the sensitivity and specificity of nucleic acid chip hybridization tests and to apparatuses suitable for carrying out said method.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP03/01972 | 2/26/2003 | WO |
