Probes and Methods for STR Genotyping

Abstract

The present invention relates to the field of DNA typing, e.g., in forensic settings. More specifically, the present invention discloses probes and methods for genotyping polymorphic short tandem repeats by relying on the fluorescence quenching properties of guanine. To this end, the degree of complementarity between an amplified DNA sample and a specifically designed probe can be assessed by measuring the fluorescence intensity of a fluorophore attached to the probe upon hybridization or melting. The probes and methods of the present invention are suitable for use with portable, inexpensive DNA analysis devices and can also be applied in fields other than forensic science, such as food fraud, diagnostics, and others.

Description

Technical Field

[0001] Technical Field of the Invention The present invention relates to the field of DNA - fingerprinting, for example, in the forensic context. More specifically, the present invention discloses probes and methods for genotyping polymorphic short tandem repeats, relying on the fluorescence quenching properties of some nucleotides on some specific fluorophores. For this purpose, the degree of complementarity between an amplified DNA sample and a specifically designed probe can be evaluated by measuring the fluorescence intensity of the fluorophore attached to the probe upon hybridization or melting. The probes and methods of the present invention are suitable for use in portable and inexpensive DNA analysis devices and can also be applied in fields other than forensic medicine, such as food adulteration, diagnosis, and others.

Background Art

[0002] Background of the Invention In a process called DNA - fingerprinting, to identify an individual based on DNA evidence, DNA polymorphisms, such as single - nucleotide polymorphisms (SNPs) or short tandem repeats (STRs), must be analyzed. The individual can represent a human, but can also represent animals and other species containing DNA.

[0003] Polymorphisms in the genome contain a vast amount of information. The most common type of genetic polymorphism is classified as an SNP. The presence of an SNP at a certain position in the genome means that within a population, the same nucleotide does not occur at this specific position in all individuals of this population. An SNP can be bi-allelic, meaning that two possible nucleotides occur at this position within a population. Recently, there has been increasing attention paid to the presence of tri- or tetra-allelic SNPs, which have greater discriminatory power than bi-allelic SNPs [1]. Mutations caused by the insertion or deletion of a single nucleotide at a certain position in the genome are sometimes also represented as SNPs but are also known as indels (short for "insertion / deletion").

[0004] Forensic DNA genotyping is today almost exclusively carried out by examining STRs. These are regions of short sequences (a few nucleotides, typically 4) that are repeated several times. STR regions are polymorphic with respect to the number of repeats and thus define the possible alleles of a certain STR region [2]. In the human genome, numerous regions containing this specific type of polymorphism are identified. By analyzing a large number of STR loci, mainly located in the non-coding regions of the human genome for forensic purposes, a statistically unique profile can be obtained. In Europe, typically, a panel of 12 STRs called the European Standard Set (ESS) was examined. This panel is now extended with 5 additional loci [3]. In the United States, the Combined DNA Index System (CODIS), which contains 13 core loci and 7 additional loci, is used [4].

[0005] After amplification by polymerase chain reaction (PCR), the number of repeats can be estimated from the length of the amplicon. This information is currently obtained almost exclusively by capillary electrophoresis, a well-known DNA separation technique. Size separation of the amplicons is achieved by applying a high potential across a capillary filled with a gel through which the amplicons migrate. Differences in electrophoretic mobility result in faster migration of shorter amplicons. These fluorescently labeled DNA fragments are detected by laser-induced fluorescence [2].

[0006] Efforts have been made to create portable versions of the tools used for CE (such as the RapidHIT system) [5], or to miniaturize this technology on a chip (such as glass) [6], yet CE still requires fairly bulky equipment. There is certainly a rapidly growing interest in implementing lab-on-a-chip (LoC) in the field of forensic DNA genotyping, due to numerous advantages such as shorter analysis times and reduced reagent consumption, as well as for high levels of parallelization and flexibility. Furthermore, the production cost of the equipment is reduced, and the ease of use for the user increases dramatically. Another major advantage is the reduced risk of contamination, especially in the field of forensic medicine. LoC combines several functions into a single device, showing a high level of integration and eliminating the need to transport samples from one device to another. Making DNA analysis portable eliminates the need to transport samples to an accredited laboratory, which also minimizes the risk of contamination and allows for faster turnaround times. Since the first hours of such an investigation are generally referred to as the "golden hour", immediate results are important for those tasked with solving the crime.

[0007] Considerable efforts have already been made to develop new assays, mainly based on hybridization. This implies that synthetically produced oligonucleotides, mainly fluorescently labeled ones, are used for STR genotyping. These techniques show several advantages in comparison with CE, for example, it is not necessary to apply a high voltage. Other problems associated with CE, namely the detection of PCR artifacts, are omitted because the probes added to the system do not hybridize with these artifacts.

[0008] A well-known example of a hybridization-based method for STR genotyping is the use of HyBeacon™ probes for melting curve analysis, as described in EP3011053A2 [7]. In this technique, only one fluorescently labeled probe per locus is used, and the number of repeats is estimated from the melting temperature of the probe [8]. This method has a number of drawbacks: i) the need for a second oligonucleotide that functions as a blocker, ii) the presence of a number of internally located fluorophores, thereby increasing the cost of the probe, and iii) probe design limitations that make it impossible to design a system that can genotype all the loci required for a complete DNA profile.

[0009] Most alternative hybridization-based techniques rely on the use of one or more fluorophores combined with a quencher moiety, thereby increasing the cost of the probe and the complexity of the system. Examples are TaqMan probes (US Patent No. 5,210,015) [9] and Molecular Beacons (US Patent No. 6150097A)

[10] . Other systems use a number of fluorophores, which also increases the cost of the probe and the complexity of the system. Examples of these systems are Scorpion probes

[11] and ECHO probes

[12] . It should be noted that most of these probes are not designed for STR genotyping.

[0010] The principle of Fluorescent Resonant Energy Transfer (FRET) between a donor and an acceptor part is often utilized in DNA-probe based methods. In most cases, both parts are attached to (one of) the oligonucleotides present in the system. Halpern et al. developed a melting curve genotyping assay relying on FRET by combining an intercalating dye and a fluorescently labeled oligonucleotide. Upon melting, the FRET between the intercalating dye and the oligonucleotide disappears and a decrease in the fluorescent signal can be observed. Since mismatches result in a lower Tm compared to perfect matches, the actual STR allele calls in the system are based on Tm detection

[13] , US12 / 276849 (

[14] ).

[0011] In the event of a mismatch, the reporter region of the probe is not expected to hybridize to the amplicon, suggesting the absence of an intercalating dye in this region, and rather a high and sharp melting curve is observed for these mismatch probes, albeit at a lower temperature. This is probably due to the presence of the intercalating dye in the remaining region of the double strand and the presence of the intercalating dye in the vicinity of single-stranded DNA. It would be beneficial if the mismatch melting curve not only occurs at a low temperature but also has a different shape (e.g., lower peak height, higher peak width). This allows for correct STR genotyping despite the presence of SNPs located in the adjacent region of the probe. A mismatch at the SNP position combined with a match in the STR region will result in a melting curve with a peak shape similar to that of a normal match probe, but at a lower temperature.

[0012] The use of intercalating dyes has several disadvantages, such as inhibition of PCR and inseparable linkages. Forensic samples often contain only very small amounts of DNA, so PCR preferably has to be performed under ideal circumstances. PCR inhibition is the reason why intercalating dyes are often added at sub-saturating concentrations, thereby increasing the risk of dye jumping. This phenomenon is related to intercalating dyes that are released from the double strand during melting but are incorporated into another double strand that has not yet melted, resulting in broadening of the melting peak. Furthermore, intercalating dyes preferentially bind to regions containing many guanines and cytosines (so-called "GC-rich regions"). This potentially results in a more prominent signal for those loci. Finally, the concentration of intercalating dyes significantly affects the melting temperature of the double strand, thereby introducing a new cause of variability in melting temperature, which is invariably detrimental when performing melting curve genotyping assays.

[15] Apart from these technical limitations, the risks to the human body associated with the use of intercalating dyes must be considered.

[0013] All of these examples suggest the need for hybridization-based genotyping assays that are, on the one hand, simpler and more robust, enabling integration in easily portable devices, and, on the other hand, providing more information than melting temperature alone. In our opinion, the simplest possible probe contains only 1 fluorophore and generates the signal based solely on interaction with the sample without the use of any other modifications (such as quenchers), other molecules (such as intercalating dyes) or even other probes (such as blockers).

[0014] Probes that are related only to the quenching properties of natural nucleotides have already demonstrated their practicality in other applications, such as species identification, qPCR, and SNP genotyping, as described by Wittwer et al. in EP2927238A1

[16] . This document describes so-called Q-probes for SNP genotyping. These probes are designed such that the fluorescently labeled nucleotide always hybridizes to the target sequence, regardless of whether the probe matches the amplicon at the SNP position. By doing so, quenching occurs during hybridization, regardless of the phenotype of the sample being examined. Since a mismatch between the probe and the sample reduces the melting temperature of the double strand, genotyping is performed by standard melting curve analysis. The quenching effect of the sample on fluorescence is not related to the fact of whether the probe and the amplicon are perfectly matched or not, and variant calling is based on one factor, the melting temperature. This has proven to be sufficiently informative for SNP calling, while for STR genotyping, a rather wide range of alleles is possible for all loci investigated, rendering the use of the probes as described by Wittwer et al. or the above-mentioned HyBeacon probes useless. STR loci have different structural properties compared to SNP loci since the possible alleles differ in length rather than in sequence only. Therefore, for all loci, an array of probes of different lengths has to be developed, together with a method to evaluate whether the probe is completely complementary to the sample, which is in stark contrast to the probes described by Wittwer et al., where only one probe is designed for one locus.

[0015] Therefore, since several structural elements (such as an anchor region and a sensor region) are essential, there is still a need to design simple probes useful for STR genotyping. SUMMARY OF THE INVENTION

[0016] Summary of the Invention The present invention relates to a plurality of probes that represent the variability of alleles at certain short tandem repeat loci within a population, each probe comprising, from 5' to 3' or from 3' to 5': 1) nucleotides that anneal to a region immediately adjacent to a specific DNA sequence of interest and that contain more nucleotides than a second adjacent region, thereby ensuring proper annealing of the sample and the probe, and thus a first adjacent region; 2) a specific DNA sequence of interest that contains at least one short tandem repeat and that anneals to the short tandem repeat region within the sample; and 3) a second adjacent region that contains at least one nucleotide and that contains at least one fluorophore, where the fluorophore is attached to a residue of the second adjacent region at a position directly complementary to a specific nucleotide that can quench the fluorophore in an efficient manner with respect to the sample, or is linked to an adjacent nucleotide either upstream or downstream of that position such that it is brought in the vicinity of one or more specific nucleotides that can quench the fluorophore in an efficient manner with respect to the sample upon hybridization of the second adjacent region with the sample, or is linked to a nucleotide two positions away either upstream or downstream of that position, comprising the plurality of probes.

[0017] The present invention further relates to the plurality of probes as described above, wherein the nucleotide is a nucleic acid analog, such as LNA. More specifically, the present invention relates to the plurality of probes as described above, wherein the fluorophore is selected from the list comprising fluorescein (FAM), hexachlorofluorescein (HEX), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), or 6-carboxytetramethylrhodamine (TAMRA). The specific nucleotide that can quench one of the fluorophores listed above is guanine.

[0018] More specifically, the present invention relates to the plurality of probes as described above, wherein the fluorophore is attached to a cytosine residue in the second adjacent region. The present invention also relates to the plurality of probes as described above, which are immobilized on a support.

[0019] The present invention also relates to a method for genotyping short tandem repeats in a sample, comprising the following steps: - Providing a sample containing DNA, - Amplifying the DNA in the sample to obtain an amplified DNA sequence, the amplified DNA sequence containing a specific DNA sequence of interest, - Obtaining a double-stranded DNA sequence of single-stranded DNA annealed to the probe by adding the plurality of probes as described above to the amplified DNA sequence, and - Denaturing the double-stranded DNA, and subsequently slowly cooling the denatured double-stranded DNA while continuously measuring the fluorescence of the fluorophore of the probe or slowly heating the double-stranded DNA while continuously measuring the fluorescence of the fluorophore of the probe, wherein a decrease or an increase in fluorescence intensity respectively provides information about whether a specific perfectly complementary short tandem repeat is present in the sample.

[0020] The present invention further relates to the method for genotyping as described above, wherein the amplification in the sample is performed by asymmetric PCR to obtain an amplified single-stranded DNA sequence. The present invention further relates to the method for genotyping as described above, wherein the amplification in the sample is performed by symmetric PCR using a biotin-labeled primer or subsequent lambda exonuclease digestion to obtain an amplified single-stranded DNA sequence. The present invention also relates to the method as described above, wherein the probe is added in solution or immobilized on a support. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

Figure 2

[0022]

Figure 3

Figure 4

Figure 5

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Figure 7

[0023]

Figure 8

Figure 9

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Figure 11

DETAILED DESCRIPTION OF THE INVENTION

[0024] Description of the Invention One aspect of the present invention relates to a probe that functions relying on the natural quenching properties of specific nucleotides for a certain fluorophore. The most common example is the fluorescein quenching effect of guanine [17, 18]. Another example is the quenching of pyrenebutyric acid by thymidine nucleotides.

[0025] The probe is defined herein as a synthetically produced oligonucleotide, some of the nucleotides of which may be modified. Examples of modifications are, by way of example, the presence of a fluorescent moiety, a molecule for attachment purposes, and the like. The probe is generally designed to interact with the molecules to be investigated and, during this interaction, the response of the probe is observed to obtain information about the molecules being investigated.

[0026] The STR genotyping probe described in the present invention consists of 3 different regions as shown in Figure 1: adjacent region 1 (FL1), a specific STR region, and adjacent region 2 (FL2). - FL1 is the region immediately adjacent to a specific DNA sequence and acts as an anchor to prevent slippage and ensure proper annealing of the sample and the probe. This implies that FL1 must be substantially longer than FL2, which is also a requirement discussed in the literature for other STR genotyping probes

[13] . If FL1 is the same length as or shorter than FL2, in the case of a mismatch, FL1 will become single-stranded and FL2 will hybridize with the sample, resulting in a signal equivalent to that generated by the matched double-strand. - The STR region is part of the polymorphism that varies between probes for a given locus. The probes are designed for all possible alleles of the locus being examined. - FL2 is substantially shorter than FL1 and is labeled at the end with a fluorophore, for example FAM. This label can be at either the 5' or 3' end. FL2 acts as a sensor and provides an indication of the degree of complementarity between the probe and the sample.

[0027] The probe is designed such that upon hybridization with a complementary amplicon, the fluorophore is brought near one or more nucleotides capable of quenching the fluorophore. In a more specific embodiment of this invention, the fluorophore is FAM, which is quenched by the presence of guanine residues. These guanine residues also have a quenching effect on other fluorophores such as HEX, TET, JOE, and TAMRA

[19] . Those skilled in the art will recognize that this is a non-limiting list. It should be noted that other combinations of fluorophores and nucleotides are also applicable for this purpose. To achieve efficient quenching of the FAM fluorophore, the fluorophore is linked to a nucleotide (mainly cytosine) at a position directly complementary to the guanine residue, or to a nucleotide adjacent (either upstream or downstream) of that position, or to a nucleotide two positions away (either upstream or downstream) from that position.

[0028] In the method described herein, a set of probes representing all possible alleles for a given STR locus is designed. The difference between complete and partial complementarity of the amplified sample and the probe can be evaluated by measuring the fluorescence intensity of the fluorophore attached to the probe during hybridization or melting. The resulting graph of fluorescence as a function of time can be divided into three parts (see Figure 2): a linear part during which the fluorescence decreases (a temperature-dependent phenomenon) and most of the probe hybridizes to the amplicon, resulting in quenching of the fluorophore; a melting part during which the fluorescence increases; and a second linear phase during which the probe becomes single-stranded. By calculating the first derivative of these graphs as a function of temperature, the melting peaks used for data interpretation are provided.

[0029] After amplification, the probe and amplicon are denatured by heating and then slowly cooled in a controlled manner to ensure correct hybridization by avoiding slippage of the probe. The probe–amplicon duplex is then melted while fluorescence is continuously measured therebetween. Upon melting, the distance between the fluorophore and the quenching guanine residue increases and the fluorescence intensity increases. This increase in fluorescence intensity occurs at a higher temperature and is more pronounced compared to mismatched combinations of the probe and amplicon when the amplicon and probe present in the PCR product share the same number of repeats. When a mismatch situation occurs, some dequenching can still be observed upon melting due to the formation of heteroduplexes. These duplexes contain mismatches in the repeat region, resulting in the formation of bulged loops. However, the melting temperature of these duplexes is lower compared to perfect complementarity and the hybridization efficiency is significantly lower: for most probes, the sensor region remains single-stranded.

[0030] The probes described herein provide information about the degree of complementarity between the probe and the sample. Information about the number of different repeats between the sample and the probe can be obtained in the case of a mismatch while speculating whether the probe and the sample have the same number of repeats. The greater the difference in the number of repeats, the lower the signal obtained. An example is given in FIG. 4 for the D8S1179 locus. The melting curves of four probes after incubation with reference sample 2800, which has alleles 14 and 15, are shown. All of the melting curves shown originate from mismatched probes. It can be clearly seen that probe 13 shows the strongest signal and probe 10 shows a weaker signal. The intensity of the signal can be defined in this example by the height of the melting peak and the melting temperature.

[0031] The unique feature of this invention is the significant amount of information that can be obtained from a number of parameters of these melting curves (Tm, peak shape, …). The melting curves result in an indication of the degree of complementarity, whereas most systems simply give a binary answer (match or mismatch). The latter systems look at only 1 parameter, for example the melting temperature or the fluorescence intensity. The unique positioning of the fluorophore, in combination with the other structural elements of the probe, makes these STR-probes highly informative. The fluorophore is positioned in a second adjacent region and thereby acts as a sensor: when the probe matches the sample, FL2 hybridizes to the sample in question. On the other hand, when the probe does not match the sample, FL2 mainly remains single-stranded or melts at a lower temperature, and the dequenching upon melting occurs less abruptly. The greater the distance between the amplicon with the quenching moiety and the fluorophore, the weaker the signal intensity. Thus, to the best of our knowledge, the STR genotyping probes discussed herein are the most elementary and informative STR genotyping probes described so far, because both the melting temperature and the fluorescence intensity are informative.

[0032] This obtained information can ultimately be analyzed in an automated way by means of artificial intelligence. Similar algorithms for high-resolution melting analysis have already been described. It is envisaged that a custom algorithm for allele calling will be developed in the future based on a large amount of data. For this purpose, the algorithm needs to be trained to call the correct allele based on the curves of samples with known alleles.

[0033] An important aspect for genotyping methods based on hybridization is the requirement of an excess of amplicon complementary to the probe. If this requirement is not met, both amplicon strands will preferentially hybridize to each other, leaving the probe single-stranded. An excess of one amplicon strand can be obtained by adapting the amplification step. After sample preparation, an amplification step should be performed to amplify the STR locus. This is typically done by means of the polymerase chain reaction (PCR), a technique well known to those skilled in the art. The region(s) to be amplified are determined by the primers used. These are short oligonucleotides complementary to sequences in the genome of the species being examined. DNA polymerase initiates amplification at the 3’ end of the primer.

[0034] In symmetric PCR, both primers are added at equal concentrations, resulting in double-stranded amplicons. When asymmetric PCR is performed, one primer is added in excess. In the first cycles, both primers are present and PCR occurs symmetrically. At some point, one primer becomes depleted, resulting in the amplification of only one of the two strands. From this point on, amplification does not occur exponentially but linearly.

[0035] Asymmetric PCR is not the only way to obtain an excess of one specific strand. After performing symmetric PCR where one of the two primers is labeled with biotin, the strand incorporating this primer can be captured by means of streptavidin beads. Another option is the specification of lambda exonuclease enzyme, which selectively degrades phosphorylated DNA strands. This modification can be introduced into one of the two primers.

[20] The above probe can also contain nucleic acid analogs, such as LNA for example. The former are non-natural constituents that are structurally similar to natural nucleic acids. Among many other examples, in particular, nucleic acids with modified bases, or modifications in the sugar component.

[0036] Example 1. Example 1: STR Genotyping Buccal Swab (D16S539 Locus) Three buccal swabs were immersed in 200 μL of sterile HPLC water. After a 30” vortex 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. The primer concentrations were 0.1 μM forward primer and 1.5 μM reverse primer. The volume of the PCR mixture was 50 μL and it contained MgCl 2+ at a concentration of 0.5 mM, 200 μM each of dNTPs, 1X Qiagen PCR buffer and 1.3 U HotStarTaq enzyme. Activation of the polymerase was done by heating the PCR mix at 95 °C for 15 minutes, followed by 60 cycles of 1 minute at 95 °C, 1 minute at 59 °C and 80 seconds at 72 °C. The primer sequences can be found in Table 1.

[0037] After asymmetric PCR, an 8.5 μL aliquot of the amplified product was dispensed into a 96-well plate. To each separate well, 1.5 μL of one specific probe was added at an initial 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 continuously measuring fluorescence using a LightCycler (Roche). The same was done during slow heating, during which the double strands melted. The probe sequences can be found in Table 1.

[0038] [Table 1] Table 1: Sequences of oligonucleotides used for the D16S539 experiment. "n" represents the number of repeats and is variable between 9 and 13.

[0039] The first derivative of the melting curve is calculated, resulting in a melting peak. The difference in melting temperature due to the difference in probe length can be investigated in this way. All samples investigated were also genotyped by conventional CE analysis as a reference.

[0040] 2. Example 2: STR Genotyping Buccal Swab (TH01 Locus) To evaluate the ability of this system to detect the slight differences in amplicon length caused by partial repeats, a melting curve experiment was performed using a probe designed for the TH01 locus. A very common allele for this locus is the 9.3 allele, which is characterized by the presence of a 10-repeat (CATT) with a T deletion in the 4th repeat. As a result, alleles 9.3 and 10 differ by only 1 nucleotide in length, which has proven to be a challenge even for CE. Two buccal swabs were extracted and amplified and analyzed in the same way as the experiment for the D16S539 locus. In contrast to the amplification for the latter locus, the forward primer was added at a concentration of 0.1 μM and the reverse primer was added at a concentration of 1.5 μM. The sequences of the primers and probes used can be found in Table 2. Sample A has alleles 9.3 and 10; sample B is homozygous (9.3:9.3).

[0041] [Table 2] Table 2: Sequences of oligonucleotides used for the TH01 experiment. "n" represents the number of repeats and is variable between 6 and 10.

[0042] 3. Example 3: STR Genotyping Reference Sample (D8S1179 Locus) To evaluate the ability of this system to detect iso-alleles caused by SNPs during repeat, a melting curve experiment was performed using a probe designed for the D8S1179 locus. The reference sample 9947a is homozygous (13:13) for the locus D8S1179, however, it is genotyped as 13:13’ by means of next-generation sequencing. The sequences corresponding to alleles 13 and 13’ can be found in Table 3. The reference sample 2800 is heterozygous (14:15) for the locus D8S1179. Both reference samples were amplified and analyzed in the same way as the experiment for the D16S539 locus. In contrast to the amplification for the latter locus, the forward primer was added at a concentration of 0.1 μM, and the reverse primer was added at a concentration of 1.5 μM. The sequences of the primers and probes used can be found in Table 3.

[0043]

Table 3-1

Table 3-2

[0044] Results 1. Example 1: STR genotyping buccal swab (D16S5339 locus) When the first derivative of the obtained melting curve was calculated, the resulting melting peaks are shown in Figs. 5-7. Fig. 5 shows the melting peaks obtained from sample 7 with alleles 9 and 12, and the allele 12 probe (P12) melts at a higher temperature compared to the allele 9 probe (P9). As shown in Fig. 5, all probes show a certain melting peak. Nevertheless, P9 and P12 display much higher peak heights and narrower peak widths. P11 shows the strongest melting peak among the mismatch probes, which follows that of the matching probe 12 in the form of being adjacent to the latter. However, the difference in Tm between P11 and P12 is still too large, suggesting non-specific annealing of P11.

[0045] Fig. 6 shows the melting curve of a homozygous sample (allele 9:9). The melting peak of the matching probe is more prominent compared to that of the heterozygous sample. Fig. 7 shows the melting curve of a heterozygous sample (alleles 11 and 13). The probe with 12 repeats is the adjacent probe of both matching probes, but still, a clear distinction can be made between the match and the mismatch. In short, sufficient information for genotyping can be inferred from hybridization or melting experiments. It should be noted that when conducting melting experiments, a slow hybridization process must precede to ensure specific annealing of the probes.

[0046] 2. Example 2: STR Genotyping Buccal Swab (TH01 Locus) It should be noted that for most of the loci examined, the matching probes show 2 peaks, while the mismatch probes show only 1. This is probably due to the presence of another allele (heterozygous sample), stutter peaks, and non-specific PCR products. The evaluation of these melting curves is, as a result, not so complicated. However, for the D16S539 locus, the matching alleles show only 1 peak, which is probably related to the shorter FL1 of those probes.

[0047] For sample A, the probe of 2 shows a melting peak at a higher temperature, and in addition to this, these peaks are characterized by a so-called "shoulder", which is actually the second peak as discussed above. The probe of 2 corresponds to the correct allele. For the homozygous sample B, only one probe shows a melting peak at a higher temperature, which corresponds to allele 9.3. Probe 10 shows a higher melting peak, but it occurs at a lower temperature and there is no shoulder peak. Thus, it can be concluded that allele 10 does not exist in the samples examined. Meanwhile, it can be concluded that the described probes and systems are capable of distinguishing between alleles 9.3 and 10.

[0048] 3. Example 3: STR Genotyping Reference Sample (D8S1179 Locus) For sample 9947a, both probes 13 and 13’ show melting peaks at higher peaks. Meanwhile, both melting peaks show a so-called shoulder, similar to the TH01 probe. Thus, it can be concluded that complementary amplicons for both probes 13 and 13’ are present in sample 9947a. For sample 2800, probes 14 and 15 show melting peaks at a higher temperature with a shoulder. Probe 14’, however, does not show a shoulder and occurs at a lower temperature. The high peak height can be explained by the presence of both allele 14 (which has the same length) and 15 (which is adjacent). It can be concluded that this method is capable of distinguishing isoalleles and is thus more informative than capillary electrophoresis.

[0049] References

Table 4-1

Table 4-2

Claims

**Claim 1** A composition comprising a number of types of probes that represent the variability of alleles at a short tandem repeat locus within a population, wherein each probe of the composition, from 5' to 3' or from 3' to 5', comprises: 1) A first adjacent region containing nucleotides that anneal to a region immediately adjacent to a specific DNA sequence of interest and contains more nucleotides than the following second adjacent region of the same probe; 2) A specific STR region that is part of the polymorphism that varies between each probe for the short tandem repeat locus and anneals to at least one short tandem repeat region contained in the specific DNA sequence of interest within the sample; and 3) A second adjacent region containing at least one nucleotide and at least one fluorophore wherein the fluorophore is: Attached to the residues of the second adjacent region at a position complementary to a specific nucleotide of the sample that has natural quenching properties for the fluorophore, or Linked to a nucleotide adjacent to the position, either upstream or downstream of the position, or linked to a nucleotide two positions away, either upstream or downstream of the position, such that upon hybridization of the second adjacent region in the sample, the fluorophore is brought into the vicinity of one or more specific nucleotides of the sample that have natural quenching properties for the fluorophore. The composition as described above. **Claim 2** The fluorophore is attached to a cytosine residue of the second adjacent region, and the specific nucleotide having natural quenching properties for the fluorophore is guanosine, or The fluorophore is pyrenebutyric acid, attached to an adenine residue of the second adjacent region, and the specific nucleotide having natural quenching properties for the fluorophore is thymidine. The composition according to claim 1. **Claim 3** The composition according to claim 1 or 2, wherein the nucleotide is a nucleic acid analog. **Claim 4** The composition according to claim 2 or 3, wherein the fluorophore quenched with guanine is selected from the list comprising fluorescein (FAM), hexachlorofluorescein (HEX), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), or 6-carboxytetramethylrhodamine (TAMRA).

5. The composition according to any one of claims 1 to 4, which is immobilized on a support.

6. A method for genotyping short tandem repeats in a sample, comprising the following steps: - providing a sample containing DNA, - amplifying the DNA in the sample to obtain an amplified DNA sequence, the sample containing a specific DNA sequence of interest, - obtaining a double-stranded DNA sequence of single-stranded DNA annealed to the probe by adding the composition according to any one of claims 1 to 5 to the amplified DNA sequence, and - denaturing the double-stranded DNA and subsequently slowly cooling the denatured double-stranded DNA while continuously measuring the fluorescence of the fluorophore of the probe or slowly heating the double-stranded DNA while continuously measuring the fluorescence of the fluorophore of the probe wherein a decrease or an increase in fluorescence intensity respectively provides information about whether a specific fully complementary short tandem repeat is present in the sample.

7. The method for genotyping according to claim 6, wherein amplifying the DNA in the sample is performed by asymmetric PCR to obtain an amplified single-stranded DNA sequence.

8. The method for genotyping according to claim 6, wherein amplifying the DNA in the sample is performed by symmetric PCR using a biotin-labeled primer or subsequent lambda exonuclease digestion to obtain an amplified single-stranded DNA sequence.

9. The method according to any one of claims 6 to 8, wherein the probe in the composition is added in solution or immobilized on a support.

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