DE69713100T2 - METHOD FOR NUCLEIC ACID ANALYSIS - Google Patents

Description

Scope of the invention

The present invention relates to nucleic acid analysis and more particularly to the determination of the binding of an oligonucleotide probe to a test nucleic acid sequence, in particular for the detection of sequence variations and the quantification of products obtained in amplification reactions.

Background of the invention

Clinical analyses of DNA sequences are typically directed at determining how a gene in a patient sample differs from a prototype normal sequence. DNA sequencing by the chain termination method developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA 1977; 74: 5463-5467) and the chemical degradation method developed by Maxam and Gilbert (Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 1977; 74: 560-564) or the use of techniques such as sequencing by hybridization (SBH) or sequencing by synthesis (see, for example, WO 93/21340) all have the potential to identify mutations and, in the same process, also reveal the consequence of the mutation at the protein coding level, etc.

Another sequencing approach is disclosed in EP-A-223 618, which describes the use of an immobilized DNA template, primers and polymerase exposed to a flow containing only one type of deoxynucleotide at a time. A downstream detection system then determines whether or not deoxynucleotide has been incorporated into the copy by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the DNA template-polymerase complex.

However, for screening purposes it is often sufficient, at least initially, to identify deviations from the normal sequence, but without directly demonstrating how a sequence differs from the normal or even roughly locating the mutation. There are a number of such techniques that speed up the analysis when compared to those involving DNA sequence determinations.

One such method uses a "label-free" detection based on surface plasmon resonance (SPR) to detect the binding of a short oligonucleotide probe to a single-stranded target sequence immobilized on a sensor chip. Because a mismatch significantly affects the binding affinity, the presence of a sequence deviation can be detected. However, this method has several disadvantages, such as that it requires the immobilization of long target sequences to the sensor chip, usually PCR products, and that the sensor chip cannot be regenerated.

Summary of the invention

The object of the present invention is to provide a method for detecting the binding of an oligonucleotide probe to a test sequence, whereby the method lacks the above-mentioned disadvantages and can be used for mismatch (mutation) detection as well as for amplification (such as PCR) product quantification.

In accordance with the present invention, the above and other objects and advantages are achieved by a method for detecting the binding of an oligonucleotide probe to a test nucleic acid sequence, comprising the steps of:

(a) providing the test nucleic acid sequence in single-stranded form,

b) bringing the test nucleic acid sequence into contact under hybridization conditions with a solution containing an oligonucleotide probe that is complementary to a defined part of a standard nucleic acid sequence,

c) immobilizing on a first solid support a nucleic acid fragment, at least a part of which is complementary to the oligonucleotide probe,

d) bringing the solution from step b) into contact with the first solid support, and

e) determining the amount of binding of the oligonucleotide probe present in the solution to its complementary nucleic acid fragment on the first solid support, wherein the amount is inversely related to the amount of binding of the oligonucleotide probe to the test nucleic acid sequence.

In a preferred embodiment, the test nucleic acid sequence is immobilized to a second solid support. In this case, the probe-containing solution is preferably separated from this second solid support before being brought into contact with the immobilized complementary nucleic acid fragment.

These and other preferred embodiments of the invention are described in more detail below.

Detailed description of the invention

The method of the invention is based on the fact that a mismatch base significantly affects the affinity of a short oligonucleotide to a complementary sequence of a single-stranded target sequence. A basic feature of the invention is that the detection is performed on oligonucleotide probe that has not bound to the target or test nucleic acid sequence, and not on probe that has bound to it. This inhibition type approach offers several advantages. First, the problem of binding of long PCR products to a sensing surface is eliminated. Second, the sensing surface will be regenerable, since the bound sample can be easily removed from the surface, in contrast to, for example, a biotin/avidin-bound PCR product. Third, since the PCR product is not bound to the sensor surface and within a preferred embodiment is bound to another solid phase, high temperature treatment can conveniently be performed to melt apart secondary structures of single-stranded DNA, as described in more detail below. Lastly, as also described in more detail below As will be described, the method allows analyses to be performed in a multispot or multi-channel format.

The term nucleic acid as used herein is to be interpreted broadly and includes DNA and RNA, including modified DNA and RNA, as well as other hybridizing nucleic acid-like molecules, such as PNA (peptide-nucleic-acid). This also applies to the term oligonucleotide probe. The size of the oligonucleotide probe is suitably within the range of, for example, from 7 to 24 bases, e.g. from 13 to 17 bases, but longer or shorter probes are also possible.

In one embodiment of the invention, the method is suitable for detecting one or more sequence deviations, such as mutations, and the approximate position thereof in a nucleic acid fragment, usually a DNA fragment. Although the method can be carried out with a single oligonucleotide probe to detect a specific mutation, it is preferred to use at least two and preferably a number of oligonucleotide probes such that the probes together cover an entire DNA sequence to be tested, such as a PCR amplified DNA sequence. The probes should then overlap by at least one base and preferably by two or three bases. Such an analysis can be carried out as follows.

Target DNA, typically PCR-amplified DNA from a patient, is bound to a solid phase. Binding to the solid phase can be achieved, as is well known in the art, for example, by including one member of a terminal specific binding pair in the PCR product and providing the other member attached to the solid phase. The PCR product can, for example, be biotinylated and the solid phase coated with avidin or streptavidin, such as streptavidin-coated magnetic beads, which are commercially available.

The bound DNA is then made single-stranded, for example by treatment with sodium hydroxide, and incubated with a mixture of short oligonucleotides, e.g. 13- 17 mers, which together would cover the entire PCR product, or a desired part of it to be analyzed, as mentioned above.

The immobilized single-stranded DNA may form a secondary structure and to prevent part or parts of the DNA sequence from thereby becoming unavailable for hybridization with the oligonucleotides, the incubation mixture is preferably heated for a suitable time, for example to 94°C, so that the secondary structures are melted apart. After cooling, the oligonucleotides present will compete and in most cases dominate the regeneration of the secondary structure.

After completion of the incubation, i.e. at equilibrium between oligonucleotide probe in solution and oligonucleotide probe bound to the immobilized PCR product, the solution is preferably separated from the solid phase. The concentrations of the respective oligonucleotide probes in the solution are then determined by contacting the solution with immobilized single-stranded DNA sequences, normally oligonucleotides complementary to the oligonucleotide probes, to hybridize the probes to the immobilized DNA and determine the amount of each probe bound to its respective complementary immobilized DNA sequence.

Any deviation of the PCR product under investigation from the wild-type sequence will result in the corresponding oligonucleotide probe having a mismatch in at least one base and thereby having a lower affinity to the PCR product, which will manifest itself as a higher concentration of the oligonucleotide in the solution compared to that in the wild-type case. The comparison with the wild-type sequence can be carried out by also performing the test described above on the wild-type sequence or, alternatively, by relating each concentration obtained to a previously prepared standard curve.

The deviation can be a point mutation, an insertion or a deletion and the specific probe that differs from the wild-type case also indicates the location of the deviation.

The above-mentioned complementary sequences to which the remaining probes in the solution are hybridized can be immobilized to separate the solid phase surfaces or to separate regions of a single surface. In the former case, the probe-containing solution is brought into contact with the different surfaces serially (e.g. by passing the solution through a number of flow cells) and in the latter case in parallel (multispot detection).

While it is preferred to immobilize the target DNA to a solid phase as described above, the analysis procedure can also be performed with the target DNA in solution. Double-stranded DNA can then be made single-stranded by heating.

Various detection principles can be used to detect the probes that bind to the immobilized complementary sequences in the last step. For example, the oligonucleotide probes can be labeled with a detectable marker, such as a chromophore or fluorophore, in which case the amount of marker can be detected by suitable photometric means, as is well known in the art. For example, the complementary sequences can be immobilized at defined positions on a chip and the markers detected with a CCD camera.

So-called marker-free detection techniques can also be used advantageously. Among such techniques are surface-sensitive detection methods, where a change in a property of a sensor surface is measured as an indication of the binding interaction on the sensor surface. Examples are mass detection methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) methods and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods.

Among the optical methods, particularly those detecting a surface refractive index can be mentioned, such as reflection optical methods, including both internal and external reflection methods, e.g. ellipsometry and attenuation wave measurement, the latter including surface plasmon resonance (SPR), Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR), attenuation wave ellipsometry, distributed total internal reflection (STIR), optical waveguide sensors, attenuation wave-based imaging, such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, etc., as well as methods based on attenuation fluorescence (TIRF) and phosphorescence. Currently attractive procedures are SPR and FTR, for which commercially available instruments are available.

In the case of mass or refractive index measuring methods, the detected hybridization response can be enhanced by binding a specific "secondary" reagent to the bound oligonucleotide probe, for example an antibody directed against an antigenic marker on the sample.

It will be readily understood that the method described above can be used to scan or screen a large number of DNA sequences for the presence of mutations. This method is also well suited for identification tests such as identification of virus or bacterial strains, HLA typing, etc.

In another embodiment of the present invention, the method is used for the quantification of amplified nucleic acid products. In order to be able to determine the amount of nucleic acids amplified by PCR (polymerase chain reaction) or related amplification methods, the sample DNA, the amount of which in the sample is unknown, is amplified together with a known amount of a "competitor" DNA of similar structure. By determining the relative amounts of amplified target and competitor DNA, the initial amount of target DNA in the sample can be determined, which is of importance for many diagnostic applications.

In accordance with the invention, the mixture of amplified target DNA and competitor DNA is immobilized to a solid phase, e.g., to magnetic streptavidin-coated beads, in the same manner as in the first embodiment described above. After single-stranding the DNA, the solid phase is then incubated with (i) an oligonucleotide probe complementary to the target DNA and (ii) an oligonucleotide probe complementary to the competitor DNA and optionally also (iii) an oligonucleotide probe complementary to a common sequence of the target DNA and the competitor DNA. The solution is then preferably first separated from the solid phase and the concentrations of the respective oligonucleotides in the solution are then determined by bringing the solution into contact with immobilized oligonucleotides that are complementary to the respective probes in the same way as in the first described embodiment. From the concentration values obtained, the amount of amplified target DNA can be determined and thereby also the amount of initial target DNA in the sample.

The above-described method of quantifying nucleic acid products from amplification reactions can, of course, be combined with the detection of sequence deviations or mutations, so that a nucleic acid fragment can be tested both qualitatively and quantitatively at the same time.

The invention will now be illustrated by the following non-limiting examples, with reference to the accompanying drawings.

Short description of the drawing

Figure 1 shows a plot of the relative response (in RE) in an SPR assay of FITC-labeled oligonucleotide remaining in solution after each incubation with complementary wild-type and mutant target, oligonucleotide bound to magnetic beads versus the concentration of complementary oligonucleotide bound to magnetic beads (in ug/ml).

Fig. 2 is a plot of the response (in RE) in the SPR assay of FITC-labeled Chlamydia-specific oligonucleotide remaining in solution after incubation with PCR-amplified Chlamydia DNA.

Examples

The analyses were performed on a BIAcore® 2000 system (Pharmacia Biosensor AB, Uppsala, Sweden) with a Sensorchip SA (Pharmacia Biosensor AB) as the optical sensor surface. The instrument was equipped with an integrated u-fluid card (IFC) that allows analysis in four cells with a single sample injection.

example 1 Detection of a sequence mutation

A model system was created based on synthetic oligonucleotides.

Magnetic streptavidin-coated beads (DynabeadsTM from Dynal, Norway) were washed according to the manufacturer’s instructions.

Two different biotinylated 17-mer oligonucleotides:

were diluted 1:2 in high ionic strength buffer (50 mM Hepes, 1 M NaCl, pH 7.4) in the range of 1- 0.00195 ug/ml. 80 μl of each dilution was incubated with 20 μl of magnetic beads for 15 minutes. Buffer without oligonucleotide was used as a control. The oligonucleotide solutions were removed and the magnetic beads were washed twice with the above buffer.

60 μl of a FITC (fluorescein isothiocyanate) labeled 13-mer oligonucleotide 100% complementary to the normal sequence was added to all tubes, with the 13-mer diluted to the concentration 0.05 μg/ml in the above buffer with the addition of 1% Tween® 20. Incubation was carried out for 15 minutes.

The solutions were removed from the magnetic beads and the beads were then washed with 20 μl of buffer containing reagent. The two resulting volumes were combined.

The normal sequence was immobilized via a streptavidin chip SA in BIAcoreTM 2000. The test solutions were injected sequentially over the surface, with intermediate amplification/increase in specificity by injection of a monoclonal antibody directed against FITC and regeneration of the surface with 100 mM NaOH. The results are shown in Table 1 below and in Fig. 1. Table 1

The results show that the 13-mer oligonucleotide binds less to the mutated sequence, which is reflected as a higher concentration in the solution.

Example 2 Detection of sequence deviations Amplification of the p53 gene exon 7

p53 gene templates from four patients (one wild type and three mutants) were subjected to PCR using the following primers 1 and 2 for the amplification of "exon 7" (365 bp) of the p53 gene:

Each amplification mixture consisted of 5 µmol of primers 1 and 2, 20 mM polymerization mix (containing the four nucleotides), 1 unit of Taq polymerase, and 5 µl of template diluted in 1 x Taq polymerase buffer. The reaction mixture was amplified in a thermocycler (GeneE, Techne) using the following program: step 1-94ºC for 10 minutes; step 2 -94ºC for 15 seconds, 58ºC for 30 seconds, 72ºC for 45 seconds; step 3-72ºC for 7 minutes. Step 2 was repeated 38 times before the procedure was completed by step 3. The product obtained with the wild-type gene is referred to below as "wild type", while the mutated genes are referred to as "Mutation 4", "Mutation 6" and "Mutation 7", respectively. The wild-type sequence for "Exon 7" as well as the positions of the above-mentioned mutations are shown in the Sequence Listing at the end of the description.

Detection of sequence deviations

Magnetic strepavidin-coated beads (DynabeadsTM from Dynal, Norway) were washed according to the manufacturer's instructions. Two tubes were then incubated with 40 µl of the "wild type" PCR product obtained as above and 40 µl of buffer (50 mM Hepes, 1 M NaCl, pH 7.4). A third tube served as a control and was incubated with buffer. The tubes were treated according to the manufacturer's instructions to make the PCR product single-stranded.

Each tube was then incubated with 60 µl of a mixture consisting of 13 FITC-labeled 13 bp oligonucleotides, shown by the lines above the respective complementary exon 7 sequence in the sequence listing and designated by the letters D, F, H, J, L, N, P, R, T, V, Y, A and "7-13", designated below as 7-d", "7-f", etc. and "7-13", respectively. During the incubation, the temperature was changed stepwise: 94ºC for one minute, and 55ºC, 45ºC, 35ºC and 29ºC for 3 minutes each. The solutions were then transferred to respective Eppendorf tubes. The respective oligonucleotides were diluted to 0.05 µg/ml in buffer containing 1% Tween® 20 diluted. The magnetic beads were then washed with 20 μl of buffer, which was mixed with the 60 μl already obtained.

Each solution was then analyzed in BIAcoreTM 2000. The biotinylated 17-mer oligonucleotides "7d-bio", "71-bio" and "7p-bio" complementary to oligonucleotides 7-d, 7-1 and 7-p above and a biotinylated oligonucleotide "Exon7-bio", Biotin-GTT CCT GCA TGG GCG GC, complementary to oligonucleotide 7-13, were immobilized in a respective channel 1 to 4 on the SA sensor chip. The analyses were then performed as described in Example 1 above.

The experiment described above was repeated for PCR products "Mutation 4", "Mutation 6" and "Mutation 7" respectively. The results are shown in Table 2 below, with responses expressed in "Resonance Units"(RE). Table 2

"Wild type" and "Mutation 4" are 100% complementary to all oligonucleotides immobilized on the sensor chip. "Mutation 6" has a central mismatch at the end of "Exon7-bio", and "Mutation 7" has a central mismatch (which reduces affinity significantly more than an end mismatch).

The expected effect of a point mutation is that a smaller amount of oligonucleotide binds to the PCR product on the solid phase, leaving more oligonucleotide in the solution being analyzed. A point mutation therefore presents itself as a higher signal for a particular oligonucleotide than that for the wild-type sequence.

The response to the wild-type sequence on "Exon7-bio" adds up to 1020 RU. Mutation 6 (end mismatch) shows an increase of approximately 300 RU, while the response for Mutation 7 (central mismatch) is enhanced by approximately 1200 RU. The increases detected therefore reflect the mutations.

Example 3 PCR product quantification

A 97 bp sequence on a plasmid containing a sequence specific for Chlamydia (Chlamydia trachomatis) was amplified by PCR. In a separate reaction, a competitor sequence contained on another plasmid was amplified. The competitor sequence differed from the first mentioned sequence in that a centrally located 17 bp Chlamydia-specific part was replaced by a 17 bp sequence taken from the lac operon. The two PCR products were then appropriate proportions (in a real case, the target and competitor templates are of course co-amplified).

Three different biotinylated oligonucleotides identical to the Chlamydia-specific sequence, the competitor sequence and a common sequence were each immobilized on a streptavidin sensor chip SA in respective channels of BIAcoreTM 2000.

Magnetic streptavidin-coated beads (DynabeadsTM from Dynal, Norway) were washed according to the manufacturer's instructions and then incubated with the PCR products of the following compositions:

The magnetic beads were washed and the DNA was made single-stranded according to the manufacturer's instructions. The beads were then incubated with a mixture of three different FITC-labeled 17-mer oligonucleotides, each complementary to one of the Chlamydia-specific sequence, the competitor sequence and a common sequence, at a concentration of 25 nM (Chlamydia-specific and competitor-specific) or 50 nM (common) for 15 minutes, after which the solution and the magnetic beads were separated.

The solutions were then injected into the BIAcoreTM 2000 instrument in series over the three measurement surfaces immobilized as described above. The response was amplified by the injection of a monoclonal antibody directed against FITC. The surfaces were regenerated by 50 mM NaOH and the next solution was then injected.

The results are shown in Fig. 2. The figure shows the anti-FITC response for the Chlamydia-specific probe after incubation with the different PCR products. The response for the common sequence was used to normalize the specific responses.

Claims (15)

Method for determining binding an oligonucleotide probe to a test nucleic acid sequence, comprising the steps of: (a) providing the test nucleic acid sequence in single-stranded form, b) bringing the test nucleic acid sequence into contact under hybridization conditions with a solution containing an oligonucleotide probe that is complementary to a defined part of a standard nucleic acid sequence, c) immobilizing a nucleic acid fragment to a first solid support, at least a part of which is complementary to the oligonucleotide probe, d) bringing the solution from step b) into contact with the first solid support, and e) determining the amount of binding of oligonucleotide probe present in the solution to its complementary nucleic acid fragment on the first solid support, the amount being inversely related to the amount of binding of the oligonucleotide probe to the test nucleic acid sequence. 2. The method of claim 1, wherein the test nucleic acid sequence is immobilized to a second solid support, wherein the solution is optionally separated from the second solid support before contacting with the immobilized nucleic acid fragment. 3. The method of claim 1 or 2, wherein the test nucleic acid sequence may contain a deviation with respect to the standard nucleic acid sequence and wherein the detected extent of binding of the oligonucleotide probe to the test nucleic acid sequence indicates whether a mismatch is present which is characterized by the presence of a deviation in the complementary part of the test nucleic acid sequence. 4. The method of claim 1, 2 or 3, further comprising performing steps a) to e) also for the standard nucleic acid sequence, and comparing the result obtained for the test nucleic acid sequence with that obtained for the standard nucleic acid sequence. 5. The method according to any one of claims 1 to 4, wherein the test nucleic acid sequence is brought into contact with a solution containing at least two different oligonucleotide probes complementary thereto, and the solution is then brought into contact with respective immobilized nucleic acid fragments complementary to the respective probes. 6. The method of claim 5, wherein probes overlap by at least one base, preferably by two or three bases. 7. The method of any one of claims 1 to 6, wherein the step of contacting the test nucleic acid sequence with the oligonucleotide probe or probes comprises temporarily heating to a temperature sufficient to melt any secondary structure of the test nucleic acid sequence. 8. Method according to one of claims 1 to 7, wherein the test nucleic acid sequence is a product of an amplification reaction, preferably the polymerase chain reaction (PCR). 9. The method of claim 8, wherein a competitor nucleic acid sequence amplified in the same amplification reaction is provided together with the test nucleic acid sequence and is contacted with a further oligonucleotide probe complementary to the competitor sequence, the amount of the additional oligonucleotide probe in the solution being determined by its binding to an immobilized complementary nucleic acid fragment and being compared with the amount of binding of the test nucleic acid sequence complementary oligonucleotide to the test nucleic acid sequence to thereby determine the original quantitative To determine the relationship between test nucleic acid sequence and competitor nucleic acid sequence. 10. The method of claim 9, wherein the test and competitor nucleic acid sequences are immobilized to a second solid support. 11. Method according to one of claims 1 to 10, wherein the binding of the oligonucleotide probe or probes to the immobilized complementary nucleic acid fragments is determined via detectable labels of the probes, preferably fluorophore or chromophore labels. 12. The method according to any one of claims 1 to 10, wherein the binding of the oligonucleotide probe or probes to the immobilized complementary nucleic acid fragments is determined by a surface-sensitive detection technique. 13. The method of claim 12, wherein the first solid support surface or surfaces are optical sensor surfaces. 14. The method of claim 13, wherein the optical sensor surface or surfaces are parts of a detector based on radio wave measurement. 15. The method of claim 14, wherein the damping wave measurement is based on surface plasmon resonance (SPR).