Hydrolysis-based probes and methods for determining STR genotypes

Abstract

The present invention relates to the field of genotyping samples containing short tandem repeat (STR) loci. More specifically, the present invention discloses a composition of matter containing an array of probes and a method for genotyping these loci that relies on RNA:DNA base pair recognition and subsequent cleavage of the RNA-containing strand. By measuring the temperature at which a DNA-RNA-DNA chimeric probe is cleaved and the resulting increase in probe fluorescence, it can be assessed whether the probe and sample share the same amount of repeats. The array of probes covers all available alleles of the STR loci being utilized and interrogated. The probes and methods of the present invention are suitable for use in portable, less expensive DNA analysis devices and can be applied in fields other than criminal investigation, such as food fraud, diagnostics, and many other fields.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to determining the genotype of samples containing short tandem repeats (STRs). The invention discloses fluorescently labeled hybrid DNA:RNA probes consisting of three DNA regions, where one region contains at least one RNA residue and another region contains at least one quencher. The invention further relates to the probe and a method utilizing the RNase H2 enzyme, which recognizes the RNA:DNA duplex formed when the probe hybridizes to a DNA sample containing STRs. The enzyme cleaves the quencher-containing region, resulting in an increased fluorescent signal. Hybridization, subsequent enzymatic recognition, and subsequent probe cleavage occur at higher temperatures when the number of repeats in the sample exactly matches the number of repeats in the probe. This probe and method are particularly useful in portable devices for forensic DNA analysis. [Background technology]

[0002] Background of the Invention Deoxyribonucleic acid (DNA) is used for personal identification purposes, such as kinship analysis and forensic DNA genotyping. Polymorphisms in DNA, such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are tested for this goal. STRs remain the polymorphism of choice for many applications. STR-locuses are characterized by short (typically tetranucleotide) repeat sequences that are polymorphic within a population in terms of repeat amount.[1]

[0003] Various regions in the human genome have been identified that contain this specific type of polymorphism. Statistically unique profiles are obtained by analyzing a large number of STR-locuses, mostly located in non-coding regions of the human genome for forensic purposes. In Europe, a panel of 12 STRs, called the European Standard Set (ESS), has typically been tested. This panel has now been expanded by the addition of five loci.[2] In the United States, the Integrated DNA Index System (CODIS), containing 13 core loci and seven additional loci, is used.[3]

[0004] Typically, these loci are analyzed using capillary electrophoresis (CE), a size separation technique for DNA. CE is a lengthy process that requires bulky equipment. Furthermore, the high potentials required for electrophoresis are related to the need for precise power supplies. In short, CE is not ideally suited to be implemented in portable devices. Standalone devices, such as the RapidHIT (Applied Biosystems) [4], are commercially available. This particular device has a mass of 82 kg, thereby hindering routine on-site analysis of DNA traces.

[0005] Criminal investigators would benefit greatly from on-site DNA testing, as this could speed up investigations. Furthermore, implementing these analyses on a chip would reduce the risk of contamination, avoid the need for highly skilled staff, and lower costs to society.[5]

[0006] Alternative detection methods for determining STR genotypes that can be incorporated into portable devices have been described. Almost all of them are hybridization-based approaches using so-called STR probes. STR loci are somewhat longer than SNP loci, which is related to the need for long probes. Hybridization-based methods rely on duplex stability. Partial mismatches between the sample and the probe, referred to herein as heteroduplex formation, will result in duplex destabilization, reflected by a lower melting temperature. However, the longer the probe, the less impact mismatches have on duplex stability. Not only are STR loci long by definition, but due to the presence of repeat units in the probe, the possible alleles have a high degree of similarity: even if the probe and sample do not share the same amount of repeats, a large fraction of the probes will perfectly match the sample, while only a small fraction will show mismatches with the sample.

[0007] To enhance the destabilizing effect of single-repeat mismatches, US9404148B2 [6,7] describes HyBeacon probes used in solution with a blocker oligonucleotide, thereby shortening the probe length. This assay was implemented in the ParaDNA device commercialized by LGC [8]. Genotyping is performed by conventional melting curve analysis. Disadvantages of this system are the limitations of the probe design, which make it impossible to design a system that can genotype all loci required for a complete DNA profile, and the need for a second oligonucleotide to function as a blocker, thereby adding a significant degree of complexity to the system. Other systems using multiple synthetic oligonucleotides are described, for example, in US9783842B

[11] , which describes a method based on differential hybridization, US7501253B2

[12] , which describes a branch migration assay, and US6753148B2, which describes a method based on the stability of the probe-sample duplex, i.e., the "sandwich hybridization method" using a capture probe and a reporter probe, and the "loop-out method"

[13] . As described for HyBeacon probes, similar drawbacks, e.g., increased complexity, are also found in the latter systems.

[0008] US12 / 276849 [9,10] describes the methodology of dpFRET, a melting curve-based approach that omits the use of blocking oligos. A drawback to this system is the use of toxic intercalating dyes, which also alter the melting behavior of the oligos. In addition to the use of multiple synthetic oligonucleotides, the introduction of an enzymatic cleavage step is an effective strategy to dramatically enhance the specificity of assays that rely on duplex destabilization.When using dpFRET methodology or other methodologies that rely on the physical distance between the probe and the sample, the melting peak is relatively broad because the signal is already generated during the annealing process.In contrast, endonucleases rely on the correct base pairing of DNA.The signal is therefore only generated after the duplex is formed, resulting in a narrower and clearer peak.

[0009] A suitable enzyme for genotyping assays is the RNase H2 enzyme, which recognizes RNA:DNA duplexes and cleaves the RNA strand. US20160130673A1

[19] describes the combined use of endonuclease activity (e.g., derived from RNase H) and exonuclease activity (e.g., derived from polymerase) for the detection of target sequences. The system is somewhat similar to TaqMan® probes, but utilizes chimeric DNA-RNA-DNA probes. The probe targets a small region of interest, e.g., a SNP or INDEL, and relies on whether the RNA region of the probe hybridizes to the target region of interest. The probe is further designed so that the mismatch is located in the center of the duplex, which is the most unstable position. Such an assay results in a binary answer (i.e., the RNA portion either hybridizes or does not), a characteristic that is ideally suited for the analysis of biallelic loci, e.g., SNP-locuses. However, such a strategy cannot be applied to STR probes, because these DNA regions are characterized by multiple possible alleles that differ in length rather than simply by sequence. In fact, the detection part of such probes may not be located in the center of the probe, but rather towards the end. Therefore, some structural adaptations to the probe (e.g., the positioning of the anchor region and RNA bases) are essential. Because the locus of interest is longer than the SNP-locus, the destabilizing effect of mismatches is reduced. This involves the fact that the RNA-portion will hybridize even when a mismatch occurs, complicating the assessment and data analysis methods.

[0010] In summary, there remains a clear need to design probes and methods for determining STR genotypes that result in high signal-to-noise, have no design limitations, and can be implemented in portable devices. [Brief explanation of the drawings]

[0011] Brief description of the figure [Figure 1] Figure 1: Probe design. The probe consists of, from 5' to 3' or 3' to 5', (i) a first flanking region that acts as an anchor to ensure correct annealing of the probe and prevent slippage; (ii) a repeat region that contains one or more repeats and includes at least one fluorescent moiety; and (iii) a second flanking region that acts as a sensor, containing at least one ribonucleotide and at least one quencher capable of quenching the fluorophore. [Figure 2] Figure 2: Probe:sample (hetero)duplex before enzymatic digestion. If the probe and sample have the same number of repeats, indicating perfect complementarity, a homoduplex will form. On the other hand, if the probe and sample do not share the same amount of repeats, a heteroduplex will form, characterized by a lower hybridization and melting temperature.

[0012] [Figure 3] Figure 3: Fluorescence upon hybridization. At high temperature, the DNA is single-stranded (denatured) and the probe remains intact. Upon cooling, the probe and sample anneal. The RNase H2 enzyme recognizes and cleaves the probe at the RNA site, causing the quencher and fluorophore to be separated from each other, which in turn causes an increase in fluorescence. Note the inverted direction of the temperature axis. [Figure 4] Figure 4: Fluorescence upon hybridization in three different situations. One sample was incubated with three different probes: a matching probe (solid line), a probe with one less repeat compared to the sample (dashed line), and a probe with one more repeat compared to the sample (dotted line). The increase in fluorescence indicates hybridization of the RNA portion. This occurs at the highest temperature for the matching probe, although the probe with one more repeat is longer and therefore has a theoretically higher melting temperature. Note the inverted direction of the temperature axis.

[0013] [Figure 5] Figure 5: Fluorescence as a function of temperature for Example 1. Matching probe 7 hybridizes at a higher temperature compared to mismatch probes 6 and 8. [Figure 6] Figure 6: First derivative of fluorescence with respect to temperature for Example 1. Matching probe 7 hybridizes at a higher temperature compared to mismatch probes 6 and 8. [Figure 7] Figure 7: First derivative of fluorescence with respect to temperature for Example 2. Matching probes 6 and 7 hybridize at a higher temperature compared to mismatch probe 8. No signal occurs for mismatch probes 9, 9.3, and 10. [Figure 8] Figure 8: First derivative of fluorescence versus temperature for Example 3. Matching probes 8 and 9.3 hybridize at higher temperatures compared to mismatch probes 6, 7, and 10. Only very limited signal occurs for mismatch probe 10. Summary of the Invention

[0014] Summary of the Invention The present invention provides the following: a) an array of oligonucleotide probes, each of which contains, from 5' to 3' or 3' to 5', the following three regions: I. a first flanking region comprising at least one nucleotide that anneals to the region immediately adjacent to the specific DNA sequence of interest and has a higher melting temperature than the second flanking region; II. A region containing a specific DNA sequence that anneals to the short tandem repeat region of interest in the sample and contains at least one fluorophore; and III. A second flanking region comprising at least two nucleotides and containing at least one ribonucleotide and at least one quencher moiety capable of efficiently quenching the fluorophore, wherein the fluorophore and quencher moiety are separated from each other by at least one ribonucleotide. Contains, and b) an RNase H2 enzyme capable of digesting the probe upon hybridization of the probe with the sample, by recognition of the RNA:DNA duplex; The present invention relates to a composition comprising:

[0015] The present invention further relates to a composition comprising an array of oligonucleotide probes as described above, wherein the quencher is attached to the 3' or 5' end of each of the probes. The present invention further relates to a composition comprising an array of oligonucleotide probes as described above, wherein the fluorophore is attached to a nucleotide of the second flanking region of each of the probes, and wherein the quencher is attached to a nucleotide of a specific DNA sequence of interest of each of the probes.

[0016] In a particular embodiment of the invention, the fluorophore is a fluorescein derivative. In a particular embodiment of the invention, the quencher is an Iowa Black FQ quencher. The present invention further relates to a composition comprising an array of oligonucleotide probes as described above, which contain more than one ribonucleotide. The present invention further relates to a composition comprising an array of oligonucleotide probes as described above, wherein the nucleotides are nucleic acid analogs. The present invention further relates to a composition comprising an array of oligonucleotide probes as described above, each of said probes being immobilized on a support.

[0017] The present invention also provides a method for determining the genotype of short tandem repeats in a sample, comprising: - providing a sample containing DNA; - amplifying DNA in said sample containing a specific DNA sequence of interest to obtain an amplified single-stranded DNA sequence; - adding an array of probes as described above to said DNA sequence to obtain a duplex of single-stranded DNA sequences annealed to said probes, adding RNase H2 enzyme, heating the mixture of sample, probe and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated; - measuring the fluorescence upon cooling of the mixture after activation of the RNase H2 enzyme, wherein an increase in fluorescence intensity provides information on whether a particular perfectly complementary short tandem repeat is present in the sample; The present invention relates to a method comprising the steps of:

[0018] The present invention further relates to a method for determining a genotype as described above, wherein said amplification in said sample is carried out by asymmetric PCR to obtain an amplified single-stranded DNA sequence. The present invention further relates to a method for determining a genotype as described above, wherein said amplification in said sample is carried out by symmetric PCR using biotin-labeled primers or by subsequent lambda exonuclease digestion to obtain an amplified single-stranded DNA sequence. The present invention further relates to a method for determining a genotype as described above, wherein said array of probes is applied in solution or immobilized onto a support.

[0019] Description of the invention The present invention relates to a composition comprising an array of oligonucleotide probes and RNase H2 enzyme. A probe is defined herein as a synthetically produced oligonucleotide consisting of two or more nucleotides and / or ribonucleotides covalently linked to each other, where some nucleotides and / or ribonucleotides may be modified. Such modifications are defined as molecules added to the oligonucleotide that do not necessarily occur in natural DNA or RNA. Examples of modifications include the presence of fluorescent moieties, quenchers, molecules for attachment purposes, melting temperature modifiers, etc. Although probes can be synthetically produced, the definition of an oligonucleotide probe herein is not limited exclusively to synthetically produced oligonucleotides. A probe is generally designed to interact with a molecule to be investigated, and upon this interaction, the response of the probe is observed and used to obtain information about the molecule to be investigated.

[0020] DNA complementarity can be explained by Chargaff's rule, which states that adenine always forms hydrogen bonds with thymine or uracil, and cytosine always forms hydrogen bonds with guanine, a process also known as Watson-Crick or Hoogsteen base pairing, resulting in double-stranded DNA. Hybridization or annealing is defined herein as the formation of a duplex or heteroduplex structure consisting of two nucleic acid strands after complementary base pairing. A duplex structure is defined as a complex of two nucleic acid strands with perfectly complementary base pairs. A heteroduplex structure is defined as a complex of two partially complementary nucleic acid strands, for example, a complex of two DNA strands extended by one or more tetranucleotide repeats.

[0021] The function of the probe array disclosed by the present invention is to determine the genotype of short tandem repeat loci (STR loci). STR loci are characterized by short (typically tetranucleotide) repeat sequences that are polymorphic within a population with respect to the amount of repeat. In contrast to single nucleotide polymorphisms (SNPs), these loci are multi-allelic, indicating that a somewhat wide range of repeat numbers occurs within a population. By determining the repeat number of enough loci, a statistically unique profile can be obtained for each individual. The term "array of probes" refers to the fact that a dedicated probe is designed for each allele of the STR locus being investigated. A probe array consists of all the different probes for a given locus. The interaction between a specific probe and a sample should be analyzed individually, and all the different probes should be physically separated, for example, using different wells of a multi-well plate or by immobilizing them in separate spots on a surface. The oligonucleotide probes disclosed by the present invention comprise a specific DNA sequence of interest 5' to 3' or 3' to 5' of a first flanking region and a second flanking region, as illustrated in Figure 1.

[0022] The first flanking region is a sequence of nucleotides and comprises at least one nucleotide. In a more convenient embodiment of the present invention, the flanking region comprises between 20 and 40 nucleotides. The first flanking region is complementary to the region immediately adjacent to the STR region, anneals to it, and ensures that the sample and the probe anneal correctly, thereby acting as an anchor. This first flanking region has a significantly higher melting temperature than the second flanking region, so hybridization initiation at the first flanking region is advantageous. To obtain correct genotype determination, it is important that the first repeat of the sample and the first repeat of the probe anneal and prevent sample slippage.

[0023] The specific DNA sequence of interest contains at least one short tandem repeat, contains at least one fluorophore, and anneals to a short tandem repeat region within a sample. In one embodiment of the present invention, the sample is DNA from which a target STR-region is amplified, for example, using the polymerase chain reaction. The second flanking region comprises at least one nucleotide, including at least one ribonucleotide, such as ATP, CTP, GTP, and UTP, and contains at least one quencher moiety capable of efficiently quenching the fluorophore.

[0024] Fluorophores are defined herein as compounds characterized by a fluorescence emission maximum between approximately 350 nm and 900 nm. A commonly used fluorescein derivative is 5-FAM (5-carboxyfluorescein). Other commonly used fluorophores include 5-hexachlorofluorescein, 6-hexachlorofluorescein, 5-tetrachlorofluorescein, 5-TAMRA (5-carboxytetramethylrhodamine), 6-TAMRA (6-carboxytetramethylrhodamine), Cy5 (indodicarbocyanine-5); Cy3 (indodicarbocyanine-3), and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

[0025] A quencher is defined as a moiety that suppresses the emission of a fluorophore when brought into close proximity to the fluorophore. A common quenching mechanism is fluorescence resonance energy transfer (FRET), but the definition of a quencher is not limited to this mechanism herein. Another mechanism, for example, is photoinduced electron transfer. Commercially available quenchers include Dabcyl, Iowa Black® FQ and RQ, ZEN®, and Black Hole Quencher, for example, BHQ-1®. In a specific embodiment of the present invention, a fluorescein derivative is used in combination with an Iowa Black FQ quencher moiety. Those skilled in the art will recognize that other combinations of fluorescent moieties and quenchers are suitable for this goal. It is crucial that the emission wavelength of the fluorophore corresponds to the optimal absorbance wavelength of the quencher. An example of a feasible fluorophore-quencher combination is Cy3 in combination with Black Hole Quencher 2.

[0026] The fluorophore and quencher moieties are separated from each other by at least one ribonucleotide. If the quencher and fluorescent moiety are linked to the same nucleotide or ribonucleotide, no signal will be generated upon digestion of the probe with a suitable enzyme, since the moieties will not be separated from each other. In a more specific embodiment of the present invention, the fluorophore and quencher are separated by 15 to 30 nucleotides. In certain embodiments of the invention, the quencher is attached to the 3' or 5' end of the probe.

[0027] The present invention further relates to an oligonucleotide probe as described above, wherein the fluorophore is attached to a nucleotide of the second flanking region and the quencher is attached to a nucleotide of the specific DNA sequence of interest. The present invention further relates to an oligonucleotide probe as described above, comprising more than one ribonucleotide. The present invention further relates to an oligonucleotide probe as described above, wherein said nucleotide is a nucleic acid analogue, such as LNA, PNA, GNA, TNA, morpholino (PMO). The probes described in this invention are functional both in solution and when immobilized on a support.

[0028] The present invention also provides the following: 1. Providing a sample containing DNA. In a more particular embodiment of the present invention, the sample contains DNA with at least one STR-locus. The source of this DNA can be human, animal, or even plant. Those skilled in the art will recognize that this is not an exclusive list. 2. Amplifying the DNA in the sample containing the specific DNA sequence of interest to obtain the amplified DNA sequence. There are several strategies for amplifying DNA, but polymerase chain reaction (PCR) is the most commonly applied method to amplify the specific sequence of interest, for example, the STR locus. In the PCR reaction, the amplified locus is determined by specifically designed primers. Amplification is carried out using a DNA polymerase enzyme. Those skilled in the art will recognize that there are many other strategies for amplifying DNA, both targeted and non-targeted. For example, isothermal DNA amplification, whole genome amplification, and rolling circle amplification. 3. Adding a probe as described above to the amplified DNA sequence to obtain a duplex of single-stranded DNA sequences annealed to the probe; 4. Addition of RNase H2 enzyme or any other enzyme capable of cleaving the probe at an RNA position when this position is hybridized to a complementary nucleotide. In a specific embodiment of the invention, 25 mU of enzyme is added. 5. Heating the mixture of sample, probe, and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated, typically 95°C, using, for example, a real-time PCR machine. 6. After activation of the RNase H2 enzyme, measure the fluorescence upon cooling the mixture, e.g., using a real-time PCR instrument. The present invention relates to a method for determining the genotype of short tandem repeats in a sample, comprising the steps of:

[0029] After activating the RNase enzyme at high temperature, the mixture is slowly cooled. In a specific embodiment of the present invention, the mixture is cooled at a rate of 0.5°C per minute. However, it should be noted that both faster and slower cooling are feasible. Upon cooling the mixture, the probe will hybridize with the amplified DNA strand of the sample. Due to the presence of the anchor region in the probe, hybridization is favored on the anchor side of the probe. This ensures that the first repeat of the probe, starting from the anchor side, will hybridize to the first repeat of the sample.

[0030] If the probe and the amplified DNA strand have exactly the same number of repeats, the perfect probe will hybridize to the sample. If the probe and the amplified DNA strand do not share the same number of repeats, heteroduplex formation will occur (Figure 2). In the latter case, hybridization will occur at a lower temperature compared to the perfect complementarity case. Because the RNase H2 enzyme is active over a wide temperature range, the probe is cleaved as soon as it hybridizes to the sample (Figure 3). As a result, the fluorescent signal of a probe with the same number of repeats as the sample increases at higher temperatures compared to a mismatched probe (Figure 4).

[0031] The present invention thus describes an STR assay that determines the hybridization temperature of a probe by enzymatic degradation. The destabilizing effect of partial mismatches between the probe and the sample is difficult to assess for STR loci, since these are, by definition, long loci. It is generally known that the longer the probe, the lower the destabilizing effect of mismatches. By introducing an enzymatic cleavage step that relies on the specific hybridization of RNA units in the probe, extremely sharp, distinct peaks are obtained, thereby optimally highlighting differences in duplex stability. Only after this specific hybridization of ribonucleotides is a signal generated, accompanied by the formation of an open-loop structure in the heteroduplex (as illustrated in Figure 2). The use of fluorescent molecules in combination with a quencher moiety results in a high signal-to-noise ratio.

[0032] The present invention further relates to a method for determining genotype as described above, wherein the amplification in the sample is carried out by asymmetric PCR to obtain an amplified single-stranded DNA sequence. Obtaining single-stranded DNA is crucial because reannealing of double-stranded amplicons will be favored over probe hybridization. Asymmetric PCR is a technique often used to obtain single-stranded DNA. To achieve this goal, primers are added to the PCR reaction mixture at various concentrations. The primer that incorporates in the strand complementary to the probe is added in excess. During the first PCR cycle, both primers are consumed, and PCR occurs exponentially. PCR will occur linearly because only the desired strand is produced upon depletion of the primer added at a lower concentration.

[0033] An alternative to asymmetric PCR is symmetric PCR, which uses biotin-labeled primers. After PCR, streptavidin beads are added to the amplified DNA. The biotin-labeled primers covalently react with streptavidin, and upon denaturation of the double-stranded amplicon, the desired strand can be isolated. Another alternative is symmetric PCR followed by lambda exonuclease digestion. Only the strand derived from the 5' phosphate-labeled primer will be digested. The present invention also relates to a method as described above, wherein said probe is added in solution or immobilized on a support. [Example]

[0034] example Example 1: Three probes (with 6, 7, and 8 repeats) designed for the TH01 locus were mixed with a synthetically produced complement with 7 repeats. The probe concentration was 0.1 μM, and the synthetic complement concentration was 1 μM. The probe sequences can be found in Table 1. After adding RNase H2 enzyme, the mixture was heated to 95°C for 10 minutes. The mixture was then slowly cooled to ensure proper hybridization of the probe and synthetic complement. Fluorescence was monitored during this hybridization phase. The first derivative of fluorescence with respect to temperature was calculated. [Table 1] Table 1: Sequences of oligonucleotides used for TH01 experiments. Ribonucleotides are preceded by an 'r'. The underlined T-nucleotide indicates fluorescein dT. Iowa Black FQ was used as the quencher.

[0035] The fluorescence decreased in all three wells, indicating that all the different probes were digested by the enzyme. However, the matching probe was digested at a higher temperature compared to the mismatched probe, indicating heteroduplex formation between the mismatched probe and the sample.

[0036] Example 2: Buccal swabs were immersed in a volume of 200 μL of sterile HPLC-water. After a 30-μL vortexing step, the swabs were removed and the water was used as input for PCR. Singleplex asymmetric PCR was performed with 30 μL of input sample. Primer concentrations were 0.1 μM forward primer and 1.5 μM reverse primer. The volume of the PCR mixture was 50 μL and contained MgCl at a concentration of 0.5 mM. 2+ The PCR mix contained 200 μM of each dNTP, 1× Qiagen PCR buffer, and 1.3 U of HotStarTaq enzyme. Polymerase activation was performed by heating the PCR mix to 95° C. for 15 minutes, followed by 60 cycles of 95° C. for 1 minute, 59° C. for 1 minute, and 72° C. for 80 seconds. Primer sequences can be found in Table 1.

[0037] After asymmetric PCR, 8.5 μL aliquots of the amplified product were divided into 96-well plates. To each separate well, 1.5 μL of specific probe was added at a starting concentration of 1 μM. These mixtures were denatured at 95°C for 10 minutes, followed by slow cooling at a ramp rate of 0.04°C / s while fluorescence was continuously measured using a LightCycler (Roche). The first derivative of the hybridization curve was calculated, resulting in the melting peak. The sequences of the probes can be found in Table 2. The samples were genotyped using CE analysis and contained alleles 6 and 7. [Table 2] Table 2:Sequence of the oligonucleotide used for the TH01 experiment. 'r' indicates that the following unit is a ribonucleotide. The underlined T-nucleotide indicates fluorescein dT. Iowa Black FQ was used as the quencher.

[0038] The first derivatives of all hybridization curves are shown in Figure 7. Significant signals could be observed for alleles 6, 7, and 8. Although probe 8 was the longest of the three probes displaying a signal, it exhibited a significantly lower hybridization temperature, indicating heteroduplex formation. The other probes showed almost no signal, indicating that the mismatches were so unstable that no hybridization occurred.

[0039] Example 3: Buccal swabs were prepared, amplified, and analyzed as described in Example 2. The same primers and probes as described in Example 2 were used. Tested samples were genotyped using CE with allele 9.3. Samples with allele 9.3 have 10 repeats but are characterized by a single nucleotide deletion in the third repeat. This is a challenging allele because hybridization of this sample with probe 10 results in a heteroduplex that is destabilized only by a single nucleotide indel (insertion / deletion).

[0040] After asymmetric PCR, 8.5 μL aliquots of the amplified product were divided into 96-well plates. 1.5 μL of probe (1 μM) was added to each well. The mixtures were denatured at 95°C for 10 minutes, followed by slow cooling at a ramp rate of 0.04°C / s while fluorescence was continuously measured using a LightCycler (Roche). The first derivative of the hybridization curve was calculated, resulting in the melting peak.

[0041] The first derivatives of all hybridization curves are shown in Figure 8. Significant signals can be observed for alleles 8 and 9.3. Probe 10, which has only one nucleotide mismatch with the positive allele 9.3, shows almost no signal. Probe 9, which is a probe adjacent to both positive alleles, and probe 7, which is a probe adjacent to the positive allele, also show melting peaks at lower temperatures compared to the positive probes. The positive probes display a second peak at a lower temperature, which may be due to the formation of a heteroduplex between probe 8 and sample 9.3, and vice versa.

[0042] [ka]

Claims

1. a) an array of oligonucleotide probes, each of said probes comprising, from 5' to 3' or 3' to 5', the following three regions in the order I, II, III: I. A first flanking region comprising a DNA sequence of between 20 and 40 nucleotides that anneals to the region immediately adjacent to the specific DNA sequence of interest and has a higher melting temperature than the second flanking region; II. A region containing a specific DNA sequence that anneals to the short tandem repeat region of interest in the sample and contains at least one fluorophore; and III. A DNA sequence comprising at least 15 nucleotides and comprising at least one ribonucleotide, and attached to at least one quencher moiety adjacent to the fluorophore that efficiently quenches the fluorophore; wherein the ribonucleotide is located three bases upstream of the quencher, wherein the fluorophore and quencher moieties are separated from each other by 15 to 30 nucleotides, including at least one ribonucleotide; the fluorophore is attached to a nucleotide of the second flanking region of each of the probes; and a second flanking region in which the fluorophore and quencher moieties are not linked to the same nucleotide or ribonucleotide; and b) an RNase H2 enzyme capable of digesting the probe upon hybridization of the probe with the sample, by recognition of the RNA:DNA duplex; and wherein the fluorophore and quencher are positioned such that an RNase H2 enzyme recognizes and cleaves the probe at the RNA position, separating the quencher and fluorophore from each other, thereby generating a signal. A composition comprising:

2. 10. A composition comprising an array of oligonucleotide probes according to claim 1, wherein a quencher is attached to the 3' or 5' end of each of the probes.

3. 3. A composition comprising an array of oligonucleotide probes according to claim 1 or 2, wherein a quencher is attached to a nucleotide of a specific DNA sequence of interest in each of the probes.

4. A composition comprising an array of oligonucleotide probes according to any one of claims 1 to 3, wherein the fluorophore is a fluorescein derivative.

5. A composition comprising an array of oligonucleotide probes according to any one of claims 1 to 4, wherein the quencher is an Iowa Black FQ quencher.

6. A composition comprising an array of oligonucleotide probes according to any one of claims 1 to 5, wherein each of the probes contains more than one ribonucleotide.

7. A composition comprising an array of oligonucleotide probes according to any one of claims 1 to 6, wherein the nucleotides are nucleic acid analogs.

8. A composition comprising an array of oligonucleotide probes according to any one of claims 1 to 7, wherein each of the probes is immobilized on a support.

9. - providing a sample containing DNA; - amplifying the DNA in said sample containing the specific DNA sequence of interest to obtain an amplified single-stranded DNA sequence; - adding an array of probes according to any one of claims 1 to 8 to a single-stranded DNA sequence to obtain a duplex of said DNA sequence annealed to the probes, - adding RNase H2 enzyme, - Heating the mixture of sample, probe and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated. - measuring the fluorescence upon cooling of the mixture after activation of the RNase H2 enzyme, wherein an increase in fluorescence intensity provides information on whether a particular perfectly complementary short tandem repeat is present in the sample; 1. A method for determining the genotype of short tandem repeats in a sample, comprising the steps of:

10. 10. The method for determining a genotype according to claim 9, wherein said amplification in a sample is carried out by asymmetric PCR to obtain an amplified single-stranded DNA sequence.

11. A method for determining a genotype as described in claim 9 or 10, wherein the amplification in the sample is carried out by symmetric PCR using biotin-labeled primers or by subsequent lambda exonuclease digestion to obtain an amplified single-stranded DNA sequence.

12. The method for determining a genotype according to any one of claims 9 to 11, wherein each of the probes is added in solution or immobilized on a support.

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