Hydrolysis-based probes and STR genotyping methods

Abstract

The present invention relates to the field of genotyping samples containing short tandem repeat (STR) loci. More specifically, the present invention discloses compositions of matter comprising probe arrays and methods for genotyping these loci that rely on recognition of RNA:DNA base pairs followed by cleavage of the RNA-containing strand. By measuring the temperature at which a chimeric DNA-RNA-DNA probe is cleaved, resulting in an increase in the fluorescence of the probe, it can be assessed whether the probe and sample share the same number of repeats. An array of probes is used that covers all possible alleles of the STR loci under investigation. The probes and methods of the present invention are well suited for use in portable, relatively inexpensive DNA analysis devices and may have applications beyond forensics, such as food fraud, diagnostics, and many other areas.

Description

Technical Field

[0001] The present invention relates to genotyping samples containing short tandem repeat sequences (STRs). The present invention discloses a fluorescently labeled hybrid DNA:RNA probe consisting of three DNA regions, one of which contains at least one RNA residue and another region contains at least one quencher. The present invention also relates to methods using the probe and an RNase H2 enzyme that recognizes the RNA:DNA duplex formed when the probe hybridizes with a DNA sample containing STRs. The enzyme cleaves the region containing the quencher, resulting in an increase in the fluorescent signal. When the number of repeats in the sample corresponds exactly to the number of repeats in the probe, hybridization, subsequent enzymatic recognition, and subsequent probe cleavage occur at higher temperatures. The probes and methods of the present invention are particularly useful in portable devices for forensic DNA analysis. Background Art

[0002] Deoxyribonucleic acid (DNA) is used for identification purposes of individuals, including kinship analysis and forensic DNA genotyping. Polymorphisms in DNA, such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are examined for this purpose. STRs remain the polymorphism of choice for many applications. STR loci are characterized by short (usually 4 nucleotide) repeat sequences that are polymorphic in the number of repeats within a given population. [1]

[0003] In the human genome, different regions containing this specific type of polymorphism are identified. Statistically unique profiles are obtained for forensic purposes by analyzing a large number of STR loci, most of which are located in the non-coding regions of the human genome. In Europe, a set of 12 STRs is usually examined, called the European Standard Set (ESS). This set has now been extended with 5 additional loci. [2] In the United States, the Combined DNA Index System (CODIS) is used, which contains 13 core loci and 7 additional loci. [3]

[0004] Typically, these loci are analyzed by capillary electrophoresis (CE), a DNA size separation technique. CE is a lengthy process that requires bulky equipment. Furthermore, the high potentials required for electrophoresis mean that an accurate power supply is required. Overall, CE is not amenable to implementation in a portable device. Stand-alone devices such as RapidHIT (Applied Biosystems) [4] are available. This particular device weighs 82 kg, which precludes routine on-site analysis of DNA traces.

[0005] Criminal investigations would benefit greatly from on-site DNA analysis, as it would speed up investigations. Among other things, performing these analyses on a chip would reduce the risk of contamination, avoid the need for highly trained personnel, and reduce costs to society.[5]

[0006] Alternative detection methods for STR genotyping that could potentially be integrated in a portable device have been described. Almost all of these methods are hybridization-based methods, using so-called STR probes. STR loci are rather long compared to SNP loci, which means that long probes are required. Hybridization-based methods rely on duplex stability. Partial mismatches between sample and probe, referred to herein as heteroduplex formation, will lead to duplex instability, which is reflected in a lower melting temperature. However, the longer the probe, the smaller the impact of mismatches on duplex stability. Due to the presence of repeating units in the probe, not only are STR loci by definition long, but also the possible alleles have a high degree of similarity: even when probe and sample do not share the same number of repeats, there is a large fraction of the probe that matches the sample perfectly, with only a small fraction showing mismatches with the sample.

[0007] To increase the destabilising effect of 1 repeat mismatch, US9404148B2[6,7] describes a HyBeacon probe used in solution together with a blocker oligonucleotide, thereby shortening the probe length. The assay is implemented in the ParaDNA device commercialised by LGC[8]. Genotyping is done by conventional melting curve analysis. The disadvantages of this system are probe design limitations that make it impossible to design a system capable of genotyping all loci required for a complete DNA profile, and the need for a second oligonucleotide as a blocker, which significantly increases the complexity of the system. Other systems using multiple synthetic oligonucleotides have been described, such as US9783842B

[11] describing a method based on differential hybridisation, US7501253B2

[12] describing a branch migration assay, and US6753148B2 describing a method based on probe and sample duplex stability, namely the "sandwich hybridisation" and "loop-out" methods using capture and reporter probes

[13] . Similar disadvantages, such as the added complexity described for the HyBeacon probes, are encountered in the latter systems as well.

[0008] US 12 / 276849 [9, 10] describes the dpFRET method, which is a melting curve based method that omits the use of blocking oligonucleotides. A disadvantage of this system is the use of toxic intercalating dyes, which also alter the melting behavior of the oligonucleotides.

[0009] In addition to the use of multiple synthetic oligonucleotides, the introduction of an enzymatic cleavage step is an effective strategy to significantly improve the specificity of assays that rely on duplex destabilization. Using the dpFRET method or any other method that relies 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 the DNA. Therefore, the signal is generated only after the duplex is formed, resulting in a narrower and more distinct peak.

[0010] An appropriate enzyme for genotyping analysis 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 (e.g. derived from a polymerase) for detecting a target sequence. The system is very similar to TaqMan® probes, but uses chimeric DNA-RNA-DNA probes. The probes target a small region of interest, such as a SNP or INDEL, and rely on whether the RNA region of the probe hybridizes with the target region of interest. The probes are further designed so that the mismatch is located in the center of the duplex, which is the least stable position. This analysis produces a binary answer (i.e., whether the RNA portion hybridizes), which is a feature that is well suited for analyzing bi-allelic loci such as SNP loci. However, this strategy cannot be applied to STR probes because these DNA regions are characterized by multiple possible alleles that differ in length rather than just sequence. In fact, the sensing portion of such probes cannot be located in the center of the probe, but rather closer to the end. Therefore, some structural adjustments to this probe, such as the positioning of the anchor region and RNA bases, are essential. Since the target locus is longer than the SNP locus, the destabilizing effect of mismatches is reduced. This means that even if a mismatch occurs, the RNA portion will hybridize, complicating the methods of evaluation and data analysis.

[0011] In summary, it is clear that there remains a great need to design STR genotyping probes and methods that yield high signal-to-noise ratios, have no design limitations, and can be implemented in portable devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 ： Probe design. The probe consists from 5' to 3' or from 3' to 5' of: (i) a first flanking region that serves as an anchor to ensure proper annealing of the probe and prevent slippage; (ii) a repeat region that comprises one or more repeats and includes at least one fluorescent moiety; (iii) a second flanking region that serves as a sensor and includes at least one ribonucleotide and at least one quencher capable of quenching the fluorophore.

[0013] Figure 2 ：Probe:sample (hetero)-duplex before enzyme digestion. Homoduplexes are formed if the probe and sample have the same number of repeats, indicating perfect complementarity. However, when the probe and sample do not share the same number of repeats, heteroduplexes will form, characterized by lower hybridization and melting temperatures.

[0014] Figure 3 ： Fluorescence during hybridization. At high temperatures, 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 separate from each other. This in turn causes an increase in fluorescence. Note the inverse direction of the temperature axis.

[0015] Figure 4 ： Fluorescence during hybridization, 3 different situations. A sample was incubated with 3 different probes: a matching probe (solid line), a probe with one less repeat than the sample (dashed line), and a probe with one more repeat than the sample (dashed line). An increase in fluorescence indicates hybridization of RNA moieties. 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 inverse direction of the temperature axis.

[0016] Figure 5 ： Fluorescence as a function of temperature, Example 1. Compared to mismatched probes 6 and 8, matched probe 7 hybridizes at a higher temperature.

[0017] Figure 6 ： First derivative of fluorescence with respect to temperature, Example 1. Compared to mismatched probes 6 and 8, matched probe 7 hybridizes at a higher temperature.

[0018] Figure 7 ： First derivative of fluorescence with respect to temperature, Example 2. Matched probes 6 and 7 hybridize at higher temperatures than mismatched probe 8. Mismatched probes 9, 9.3 and 10 show no signal.

[0019] Figure 8 ： First derivative of fluorescence with respect to temperature, Example 3. Matched probes 8 and 9.3 hybridize at higher temperatures than mismatched probes 6, 7 and 10. Mismatched probe 10 shows only very limited signal. Summary of the invention

[0020] The present invention relates to a composition comprising:

[0021] a) An oligonucleotide probe array, wherein each of the probes comprises the following three regions from 5' to 3' or from 3' to 5':

[0022] I. a first flanking region comprising at least one nucleotide which anneals to a region directly adjacent to the target specific DNA sequence and has a higher melting temperature than the second flanking region,

[0023] II. a region comprising a specific DNA sequence that anneals to a target short tandem repeat region within the sample and which comprises at least one fluorophore, and

[0024] III. a second flanking region comprising at least 2 nucleotides, and comprising at least one ribonucleotide and at least one quencher moiety capable of effectively quenching the fluorophore, wherein the fluorophore and the quencher moiety are separated from each other by at least one ribonucleotide, and

[0025] b) RNase H2 enzyme, which is capable of digesting the probe by recognizing RNA:DNA duplexes when the probe is hybridized to the sample.

[0026] The present invention also 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.

[0027] The present invention also 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 target specific DNA sequence of each of the probes.

[0028] In a specific embodiment of the invention, the fluorophore is a fluorescein derivative.

[0029] In a specific embodiment of the invention, the quencher is Iowa Black Fq quencher.

[0030] The present invention also relates to a composition as described above comprising an oligonucleotide probe array comprising more than one ribonucleotide.

[0031] The present invention also relates to a composition comprising an oligonucleotide probe array as described above, wherein the nucleotides are nucleic acid analogs.

[0032] The present invention also relates to a composition comprising an array of oligonucleotide probes as described above, wherein each of said probes is immobilized on a support.

[0033] The present invention also relates to a method for genotyping a short tandem repeat sequence in a sample, comprising the following steps:

[0034] - provide a sample containing DNA,

[0035] - amplifying the DNA containing the target specific DNA sequence in the sample to obtain an amplified single-stranded DNA sequence,

[0036] - adding a probe array as described above to said DNA sequence to obtain a duplex of single-stranded DNA sequences annealed to said probes,

[0037] - Add RNase H2 enzyme,

[0038] - Heat the mixture of sample, probe and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated,

[0039] - Fluorescence is measured upon cooling the mixture after activation of the RNase H2 enzyme, wherein the increase in fluorescence intensity provides information on whether a specific, fully complementary short tandem repeat sequence is present in the sample.

[0040] The present invention also relates to the genotyping method as described above, wherein said amplification within said sample is performed by asymmetric PCR to obtain amplified single-stranded DNA sequences.

[0041] The present invention also relates to a genotyping method as described above, wherein said amplification within said sample is performed by symmetric PCR using biotin-labeled primers or subsequent lambda exonuclease digestion to obtain amplified single-stranded DNA sequences.

[0042] The present invention also relates to a genotyping method as described above, wherein the probe array is added to a solution, or is immobilized on a support.

[0043] Description of the invention

[0044] The present invention relates to compositions, and the compositions comprise oligonucleotide probe arrays and RNase H2 enzymes.Probes are defined herein as synthetically manufactured oligonucleotides, consisting of 2 or more nucleotides and / or ribonucleotides covalently linked to each other, some of which can be modified.This modification is defined as a molecule attached to an oligonucleotide, which is not necessarily present in natural DNA or RNA.Examples of modifications are, for example, the presence of a fluorescent moiety, the presence of a quencher, the presence of a molecule for attachment purposes, a modifier of a melting temperature, etc.Probes can be synthetically manufactured, but the definition of oligonucleotide probes is not limited to specially synthetically manufactured oligonucleotides in this article.Probes are usually designed in a way that they will interact with the molecules being studied, and the response of the probe to this interaction will be observed and used to obtain information about the molecules being studied.

[0045] Chargaff's rules explain the complementarity of DNA, stating that adenine always forms hydrogen bonds with thymine or uracil, while cytosine 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 completely complementary base-paired nucleic acid strands. A heteroduplex structure is defined as a complex of two partially complementary nucleic acid strands, such as two DNA strands that are delayed by one or more 4-nucleotide repeats.

[0046] The function of the probe array disclosed in the present invention is the genotyping of short tandem repeat loci (STR-locus). STR loci are characterized by short (usually 4 nucleotides) repeat sequences that are polymorphic in a specific population with respect to the number of repeats. Compared to single nucleotide polymorphisms (SNPs), these loci are multi-allelic, indicating that there is a fairly wide range of repeat numbers in the population. By determining the number of repeats for enough loci, a statistically unique profile of an individual is obtained. The wording "probe array" means that for each allele of the STR locus under investigation, a dedicated probe is designed. The probe array consists of all different probes for a certain locus. The interaction between a specific probe and a sample should be analyzed separately, which means that all different probes should be physically separated, for example by different wells on a multi-well plate, or by fixing them at different points on a surface.

[0047] like Figure 1 As shown, the oligonucleotide probe disclosed in the present invention comprises a first flanking region, a specific target DNA sequence and a second flanking region from 5' to 3' or from 3' to 5'.

[0048] The first flanking region is a nucleotide sequence and comprises at least one nucleotide. In a more convenient embodiment of the invention, the flanking region comprises 20 to 40 nucleotides. The first flanking region is complementary to and anneals with the region immediately adjacent to the STR region and ensures proper annealing of the sample and the probe, thus serving as an anchor. Since the first flanking region has a significantly higher melting temperature than the second flanking region, the initiation of the hybridization is privileged at the first flanking region. In order to obtain correct genotyping, it is essential that the first repeat of the sample anneals with the first repeat of the probe and prevents sample slippage.

[0049] The target specific DNA sequence comprises at least one short tandem repeat sequence and comprises at least one fluorophore and anneals to the short tandem repeat region in the sample. In one embodiment of the present invention, the sample is DNA, wherein the target STR region is amplified by, for example, polymerase chain reaction.

[0050] The second flanking region comprises at least one nucleotide and comprises at least one ribonucleotide, such as ATP, CTP, GTP and UTP, and contains at least one quencher moiety capable of effectively quenching the fluorophore.

[0051] Fluorophores are defined herein as compounds characterized by a fluorescence emission maximum between about 350 nm and 900 nm. A commonly used fluorescein derivative is 5-FAM (5-carboxyfluorescein). Other commonly used fluorophores are 5-hexachloro-fluorescein, 6-hexachloro-fluorescein, 5-tetrachloro-fluorescein, 5-TAMRA (5-carboxytetramethylrhodamine), 6-TAMRA (6-carboxytetramethylrhodamine), Cy5 (indole dicarbocyanine-5); Cy3 (indole dicarbocyanine-3) and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionic acid).

[0052] A quencher is defined as a moiety that inhibits the emission of a portion of a fluorophore when in proximity to the fluorophore. A common quenching mechanism is fluorescence resonance energy transfer (FRET), but the definition of a quencher herein is not limited to this mechanism. Other mechanisms are, for example, photoinduced electron transfer. Commercially available quenching groups include: Dabcyl, Iowa Black ® FQ and RQ, ZEN™ and Black Hole quenchers such as BHQ-1 ® .

[0053] In a specific embodiment of the invention, a fluorescein derivative is used in combination with an Iowa Black FQ quencher moiety. One skilled in the art will recognize that other combinations of fluorescent moieties and quenchers are suitable for this purpose. It is critical that the emission wavelength of the fluorophore corresponds to the optimal absorption wavelength of the quencher. An example of a possible combination of a fluorophore and a quencher is Cy3 and Black Hole quencher 2.

[0054] The fluorophore and quencher moieties are separated from each other by at least one ribonucleotide. If the quencher and the fluorescent moiety are attached to the same nucleotide or ribonucleotide, no signal will appear when the probe is digested by a suitable enzyme because the two moieties will not separate from each other. In a more specific embodiment of the invention, the fluorophore and the quencher are separated by 15 to 30 nucleotides.

[0055] In specific embodiments of the invention, the quencher is attached to the 3' or 5' end of the probe.

[0056] The present invention also relates to an oligonucleotide probe as described above, wherein the fluorophore is attached to the nucleotides of the second flanking region, and wherein the quencher is attached to the nucleotides of the target specific DNA sequence. The present invention further relates to an oligonucleotide probe as described above, which comprises more than one ribonucleotide.

[0057] The present invention also relates to an oligonucleotide probe as described above, wherein the nucleotide is a nucleic acid analogue, such as LNA, PNA, GNA, TNA, morpholino (PMO).

[0058] The probes described in the present invention are functional both in solution and immobilized on a support.

[0059] The present invention also relates to a method for genotyping a short tandem repeat sequence in a sample, comprising the following steps:

[0060] 1. Provide a sample containing DNA. In a more specific embodiment of the present invention, the sample contains DNA having 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 a limiting list.

[0061] 2. Amplify the DNA in the sample containing the target specific DNA sequence to obtain an amplified DNA sequence. There are many strategies for amplifying DNA, however, polymerase chain reaction (PCR) is the most commonly used method to amplify specific target sequences such as STR loci. In the PCR reaction, the amplified loci are determined by specially designed primers. Amplification is performed using a DNA polymerase. Those skilled in the art will recognize that there are many other strategies for amplifying DNA, including targeted or non-targeted. Examples are isothermal DNA amplification, whole genome amplification, and rolling circle amplification.

[0062] 3. Adding the probe as described above to the amplified DNA sequence to obtain a duplex of the single-stranded DNA sequence annealing to the probe.

[0063] 4. Add RNase H2 enzyme or any other enzyme that can cleave the probe at the RNA position when it is hybridized to the complementary nucleotide. In a specific embodiment of the present invention, 25 mU of enzyme is added.

[0064] 5. Heat the mixture of sample, probe and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated, usually 95°C, using, for example, a real-time PCR instrument.

[0065] 6. Measure fluorescence upon cooling the mixture after activation of the RNase H2 enzyme, using, for example, a real-time PCR instrument.

[0066] 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 faster and slower cooling are both feasible. After the mixture is cooled, the probe will hybridize with the amplified DNA strand in the sample. Due to the presence of the anchor region in the probe, hybridization is performed preferentially on the anchor side of the probe. This ensures that the first repeat of the probe, starting from the anchor side, will hybridize with the first repeat of the sample.

[0067] If the probe and the amplified DNA strand have exactly the same number of repeats, the intact probe will hybridize to the sample. When the probe and the amplified DNA strand do not have the same number of repeats, a heteroduplex will be formed ( Figure 2 In the latter case, hybridization will occur at a lower temperature than in the case of perfect complementarity. Since the RNase H2 enzyme is active over a wide temperature range, the probe will be cleaved immediately after hybridization with the sample ( Figure 3 ). Therefore, the fluorescence signal of the probe with the same number of repeats as the sample will increase at higher temperatures compared to the mismatched probe ( Figure 4 ).

[0068] Thus, the present invention describes an STR assay in which the probe hybridization temperature is determined by enzymatic digestion. For STR loci, the destabilizing effect of partial mismatches between probe and sample is difficult to assess, since these loci are defined as long loci. It is well 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 and distinct peaks are obtained, thereby optimally highlighting the differences in duplex stability. Only after this ribonucleotide-specific hybridization is an open ring structure (e.g., Figure 2 The combination of fluorescent molecules and quencher moieties results in a high signal-to-noise ratio.

[0069] The present invention also relates to a genotyping method as described above, wherein the amplification in the sample is performed by asymmetric PCR to obtain amplified single-stranded DNA sequences. It is essential to obtain single-stranded DNA because reannealing of double-stranded amplicons is more favorable than probe hybridization. Asymmetric PCR is a commonly used technique for obtaining single-stranded DNA. To achieve this, primers are added to the PCR reaction mixture at different concentrations. Primers that will be incorporated into the strand complementary to the probe will be added in excess. In the first PCR cycle, both primers will be consumed and PCR will grow exponentially. After the primers added at a lower concentration are exhausted, PCR will occur linearly because only the desired strand is produced.

[0070] An alternative to asymmetric PCR is symmetric PCR using biotin-labeled primers. After PCR, streptavidin beads are added to the amplified DNA. The biotin-labeled primers will covalently react with streptavidin, and after denaturation of the double-stranded amplicon, the desired strand can be separated. Another option is symmetric PCR and subsequent lambda exonuclease digestion. Only the strand originating from the 5' phosphate-labeled primer will be digested.

[0071] The present invention also relates to a method as described above, wherein the probe is added to a solution, or is immobilized on a support. DETAILED DESCRIPTION

[0072] Embodiment 1:

[0073] 3 different probes designed for the TH01 locus (with 6, 7 and 8 repeats) were mixed with a synthetically manufactured complement with 7 repeats. The probe concentration was 0.1 µM and the synthetic complement concentration was 1 µM. The probe sequences are shown in Table 1. After the addition of RNaseH2 enzyme, the mixture was heated to 95°C for 10 minutes. Thereafter, the mixture was slowly cooled to ensure proper hybridization of the probe and the synthetic complement. During this hybridization phase, fluorescence was monitored. The first derivative of fluorescence with respect to temperature was calculated.

[0074]

[0075] Table 1: Oligonucleotide sequences used for TH01 experiments. Ribonucleotides are preceded by an "r". Underlined T-nucleotides represent fluorescein dT. Iowa Black FQ was used as a quencher.

[0076] The fluorescence in all three wells decreased, indicating that all the different probes were digested by the enzyme. However, the matched probes were digested at a higher temperature than the mismatched probes. This indicates that the mismatched probes and the sample formed a heteroduplex.

[0077] Embodiment 2:

[0078] Buccal swabs were immersed in 200 µL sterile HPLC-water. After a 30” vortexing step, the swabs were removed and the water was used as input for PCR. Singleplex asymmetric PCR was performed using 30 µL of input sample. Primer concentrations were 0.1 µM forward primer and 1.5 µM reverse primer. The volume of the PCR mix was 50 µL and contained 0.5 mM MgCl 2+, 200 µM each of dNTPs, 1× Qiagen PCR buffer, and 1.3 U of HotStarTaq enzyme. Activation of the polymerase was accomplished by heating the PCR mixture at 95°C for 15 min, followed by 60 cycles of 95°C for 1 min, 59°C for 1 min, and 72°C for 80 s. Primer sequences are shown in Table 1.

[0079] After asymmetric PCR, 8.5 µL aliquots of the amplified product were dispensed in 96-well plates. In each individual well, 1.5 μL of one specific probe was added at a starting concentration of 1 μM. These mixtures were denatured at 95°C for 10 min and then slowly cooled 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 to obtain the melting peak. The probe sequences are shown in Table 2. The samples were genotyped using CE analysis and had alleles 6 and 7.

[0080]

[0081] Table 2: Oligonucleotide sequences used for TH01 experiments. "r" indicates that the following units are ribonucleotides. Underlined T-nucleotides represent fluorescein dT. Iowa Black FQ was used as a quencher.

[0082] The first derivative of all hybridization curves is Figure 7 As shown. Clear signals can be observed for alleles 6, 7, and 8. Although probe 8 is the longest of the three probes that show signals, it shows a significantly lower hybridization temperature, indicating heteroduplex formation. The other probes show almost no signal, indicating that the mismatches are too unstable to hybridize.

[0083] Embodiment 3:

[0084] Buccal swabs were prepared, amplified, and analyzed as described in Example 2. The same primers and probes were used as described in Example 2. The samples examined were genotyped using CE with allele 9.3. The sample with allele 9.3 had 10 repeats, but was characterized by a 1 nucleotide deletion in its 3rd repeat. These are challenging alleles because hybridization of this sample with probe 10 resulted in a heteroduplex that was unstable due to only a single nucleotide insertion / deletion (indel).

[0085] After asymmetric PCR, 8.5 µL aliquots of the amplified product were dispensed in 96-well plates. 1.5 µL of probe (1 µM) was added to each individual well. These mixtures were denatured at 95 °C for 10 min and then slowly cooled 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 to obtain the melting peak.

[0086] The first derivative of all hybridization curves is Figure 8 As shown. Obvious signals of alleles 8 and 9.3 can be observed. Probe 10 has only 1 nucleotide mismatch with the positive allele 9.3 and shows almost no signal. Probe 9 is an adjacent probe for the two positive alleles, and probe 7 is also an adjacent probe for the positive allele, showing a melting peak at a lower temperature compared to the positive probe. The positive probe shows a second peak at a lower temperature, which can be used to form a heteroduplex between probe 8 and sample 9.3, and vice versa.

[0087] References

[0088] 1.Butler, JM, Forensic DNA typing: biology, technology, and genetics of STR markers. 2005: Elsevier.

[0089] 2.Schneider, PM, Expansion of the European standard set of DNAdatabase loci-the current situation. Profiles in DNA, 2009. 12: p. 6-7.

[0090] 3.Hares, DR, Selection and implementation of expanded CODIS coreloci in the United States. Forensic Science International: Genetics, 2015.17: p. 33-34.

[0091] 4.Hennessy, L.K., et al., Developmental validation studies on theRapidHIT™ human DNA identification system. Forensic Science International:Genetics Supplement Series, 2013. 4(1): p. e7-e8.

[0092] 5.Bruijns, B., et al., Microfluidic devices for forensic DNAanalysis: A review. Biosensors, 2016. 6(3): p. 41.

[0093] 6.Gale, N., et al., Oligonucleotides and uses thereof. 2016, GooglePatents.

[0094] 7.Gale, N., et al., Rapid typing of STRs in the human genome byHyBeacon® melting. Organic & biomolecular chemistry, 2008. 6(24): p. 4553-4559.

[0095] 8.Blackman, S., et al., Developmental validation of the ParaDNA®Intelligence System—A novel approach to DNA profiling. Forensic ScienceInternational: Genetics, 2015. 17: p. 137-148.

[0096] 9.Halpern, M. and P.M. Ellis, Dye probe fluorescence resonance energytransfer genotyping. 2010, Google Patents.

[0097] 10.Halpern, M.D. and J. Ballantyne, An STR melt curve genotypingassay for forensic analysis employing an intercalating dye probe FRET.Journal of forensic sciences, 2011. 56(1): p. 36-45.

[0098] 11.Mamone, J., M.N. Feiglin, and H. Gamper, STR genotyping bydifferential hybridization. 2017, Google Patents.

[0099] 12.Pourmand, N., R.W. Davis, and S.X. Wang, DNA fingerprinting usinga branch migration assay. 2009, Google Patents.

[0100] 13.Sosnowski, R.G. and E. Tu, Methods and apparatus for detectingvariants utilizing base stacking. 2004, Google Patents.

[0101] 14.Moghaddam, P.P. and F.J. Herrmann, Randomized full-waveforminversion: a dimenstionality-reduction approach, in SEG Technical ProgramExpanded Abstracts 2010. 2010, Society of Exploration Geophysicists. p. 977-982.

[0102] 15.Tsuchiya, T., Method of detecting mismatching regions. 2007,Google Patents.

[0103] 16.Kemp, J., et al., DNA profiling and SNP detection utilizing microarrays. 2006, Google Patents.

[0104] 17.Walder, JA, et al., RNase H-based assays utilizing modified RNAmonomers. 2014, Google Patents.

[0105] 18. Walder, JA, J. Dobosy, and MA Behlke, Cleavable hairpinprimers. 2018, Google Patents.

[0106] 19.Li, J., et al., Nucleic acid detection by oligonucleotide probescleaved by both exonuclease and endonuclease. 2016, Google Patents. Sequence Listing <110> Ghent University <120> Hydrolysis-based probes and STR genotyping methods <130> P2019 / 085 PCT sequence listing <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 52 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 1 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcacca tg 52 <210> 2 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 2 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc accatg 56 <210> 3 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 3 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc attcaccatg 60 <210> 4 <211> 75 <212> DNA <213> Artificial sequence <220> <223> Complementary to the probe <400> 4 acagactcca tggtgaatga atgaatgaat gaatgaatga gggaaataag ggaggaacag 60 gccaatggga atcac 75 <210> 5 <211> 79 <212> DNA <213> Artificial sequence <220> <223> Complementary to the probe <400> 5 acagactcca tggtgaatga atgaatgaat gaatgaatga atgagggaaa taagggagga 60 acaggccaat gggaatcac 79 <210> 6 <211> 83 <212> DNA <213> Artificial sequence <220> <223> Complementary to the probe <400> 6 acagactcca tggtgaatga atgaatgaat gaatgaatga atgaatgagg gaaataaggg 60 aggaacaggc caatgggaat cac 83 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 gtgattccca ttggcctgtt c 21 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 8 attcctgtgg gctgaaaagc tc 22 <210> 9 <211> 52 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 9 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcacca tg 52 <210> 10 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 10 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc accatg 56 <210> 11 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 11 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc attcaccatg 60 <210> 12 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 12 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc attcaccatg 60 <210> 13 <211> 67 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 13 ctgttcctcc cttatttccc tcattcattc atcattcatt cattcattca ttcattcatt 60 caccatg 67 <210> 14 <211> 64 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 14 ctgttcctcc cttatttccc tcattcattc attcattcat tcattcattc attcattcac 60 catg 64

Claims

1. A composition comprising: a) An oligonucleotide probe array, wherein each probe comprises the following three regions directly linked in the order of I, II and III from 5' to 3' or from 3' to 5': I. a first flanking region that is complementary to and anneals to a region immediately adjacent to one end of a target short tandem repeat region within a sample and has a higher melting temperature than a second flanking region, wherein the first flanking region is a DNA sequence comprising 20 to 40 deoxyribonucleotides, II. A region consisting of a specific DNA sequence comprising at least one short tandem repeat sequence and at least one fluorophore, wherein the short tandem repeat sequence anneals to a target short tandem repeat region within the sample, and III. a second flanking region that is complementary to a region immediately adjacent to the other end of the target short tandem repeat region within the sample, the second flanking region comprising at least 2 nucleotides and comprising at least one ribonucleotide and at least one quencher capable of effectively quenching the fluorophore, wherein the fluorophore and the quencher are separated from each other by 15 to 30 nucleotides in which there is at least one ribonucleotide; and b) RNase H2 enzyme, which is capable of digesting the probe by recognizing RNA:DNA duplexes when the probe is hybridized to the sample. 2 . The composition of claim 1 , wherein the quencher is attached to the 3′ or 5′ end of each probe.

3. The composition of claim 1 or 2, wherein the quencher is attached to a nucleotide in the second flanking region of each probe, and wherein the fluorophore is attached to a deoxyribonucleotide of a specific DNA sequence of each probe.

4. The composition of claim 1 or 2, wherein the fluorophore is a fluorescein derivative.

5. The composition of claim 1 or 2, wherein the quencher is Iowa Black FQ quencher.

6. The composition of claim 1 or 2, wherein each probe comprises more than one ribonucleotide.

7. The composition of claim 1 or 2, wherein the nucleotides are nucleic acid analogs.

8. The composition according to claim 1 or 2, wherein each probe is immobilized on a support.

9. Use of the composition of any one of claims 1 to 8 in the preparation of a kit for a method for genotyping short tandem repeat sequences in a sample, the method comprising the following steps: - provide a sample containing DNA, - amplifying the DNA containing the target short tandem repeat region in the sample to obtain an amplified single-stranded DNA sequence, - adding the oligonucleotide probe array as claimed in any one of claims 1 to 8 to the single-stranded DNA sequence to obtain a duplex of the single-stranded DNA sequence annealed to the probes in the oligonucleotide probe array, - Add RNase H2 enzyme, - Heat the mixture of sample, probe and RNase H2 enzyme to a temperature at which the RNase H2 enzyme is activated, - Fluorescence is measured upon cooling the mixture after activation of the RNase H2 enzyme, wherein the increase in fluorescence intensity provides information on whether a specific, fully complementary short tandem repeat sequence is present in the sample.

10. The use according to claim 9, wherein the amplification is performed by asymmetric PCR to obtain an amplified single-stranded DNA sequence.

11. The use according to claim 9, wherein the amplification is performed by symmetric PCR using biotin-labeled primers and subsequent lambda exonuclease digestion to obtain amplified single-stranded DNA sequences.

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