Patent Description:
Deoxyribonucleic acid (DNA) is used for identification purposes of individuals, including kinship analysis and forensic DNA genotyping. Polymorphisms in the DNA, e.g. Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) are examined for this goal. STRs remain the polymorphism of choice for many applications. STR-loci are characterised by short (typically <NUM> nucleotides) repeating sequences that are polymorphous within a certain population regarding the amount of repeats.

In the human genome, different regions containing this specific type of polymorphism are identified. A statistically unique profile is obtained by analysing a large number of STR-loci, mostly located in the noncoding regions of the human genome for forensic purposes. In Europe, typically a panel of <NUM> STRs was examined, called the European Standard Set (ESS). This panel is now expanded with <NUM> additional loci. [<NUM>] In the US, the Combined DNA Index System (CODIS) is used, containing <NUM> core loci and <NUM> additional loci.

Typically, these loci are analysed by means of capillary electrophoresis (CE), a DNA size separation technique. CE is a lengthy process that requires bulky equipment. Moreover, the high potential needed for the electrophoresis implicates the need for an accurate power supply. Altogether, CE is not ideally suited to be implemented in a portable device. Standalone devices, e.g. the RapidHIT (Applied Biosystems) [<NUM>] are on the market. This particular device has a mass of <NUM>, thereby hampering the on-site analysis of DNA traces in the routine.

Crime investigations would benefit tremendously from on-site DNA analysis, as this could speed up the inquiry. Besides that, implementation of these analyses on a chip would reduce the risk for contamination, avoid the need for highly trained staff and lower the cost for the society.

Alternative detection methods for STR genotyping that could potentially be integrated in a portable device have been described. Almost all of them are hybridisation based approaches, using so-called STR probes. STR loci are rather long compared to SNP loci, which implicates the need for long probes. Hybridisation based methods rely on duplex stability. A partial mismatch between sample and probe, herein called hetero-duplex formation, will result in destabilisation of the duplex, reflected by a lower melting temperature. However, the longer a probe is, the lower the impact of a mismatch on duplex stability is. Not only are STR-loci by definition long, the possible alleles have a high degree of similarity, due to the presence of repeating units in the probe: even when a probe and a sample do not share the same amount of repeats, there is a large fraction of the probe that matches the sample perfectly, with only a small fraction showing a mismatch with the sample.

In order to increase the destabilising effect of a <NUM>-repeat mismatch, <CIT> [<NUM>, <NUM>] describes the HyBeacon probes that are used in solution, along with a blocker oligonucleotide, thereby shortening the probe length. This assay was implemented in the ParaDNA device, commercialised by LGC [<NUM>]. Genotyping is done by traditional melting curve analysis. Drawbacks to this system are probe design restrictions, making design of a system capable of genotyping all the loci needed for a complete DNA profile impossible, and the need for a second oligonucleotide functioning as a blocker, thereby adding a significant degree of complexity to the system. Other systems using multiple synthetic oligonucleotides are described, e.g. <CIT> [<NUM>] which describes a method based on differential hybridisation, <CIT> [<NUM>] which describes a branch migration assay and <CIT> which describes methods based on the stability of probe and sample duplex, namely a 'sandwich hybridisation method' using a capture probe and a reporter probe and a 'loop-out method' [<NUM>]. Similar drawbacks, e.g. the increase of complexity, as described for the HyBeacon probes are also encountered in the latter systems.

<CIT> [<NUM>, <NUM>] describes the dpFRET methodology, which is a melting curve based approach omitting the use of blocking oligos. Drawback to this system is the use of a toxic intercalating dye, which also alters the melting behaviour of oligos.

Besides the use of multiple synthetic oligonucleotides, the introduction of an enzymatic cleavage step is a valid strategy to enhance the specificity of an assay relying on duplex destabilisation dramatically. Using the dpFRET methodology or any other methodology relying on the physical distance between probe and sample, melting peaks are relatively broad as a signal is already being generated during the process of annealing. On the contrary, an endonuclease relies on the correct basepairing of DNA. A signal is therefore only generated after duplex formation, resulting in more narrow and distinctive peaks.

A suitable enzyme for genotyping assays is the RNase H2 enzyme, which recognises an RNA:DNA duplex and cleaves the RNA strand. <CIT> [<NUM>] describes the combined use of endonuclease activity (e.g. originating from RNase H) and exonuclease (e.g. originating from a polymerase) for detection of a target sequence. Said system is rather similar to TaqMan® probes but makes use of a chimeric DNA-RNA-DNA probe. The probe targets small regions of interest, e.g. a SNP or an INDEL and relies on whether or not the RNA-region of the probe hybridises to the target region of interest. The probes are further designed in such a way that the mismatch is positioned in the centre of the duplex, which is the most destabilizing position. Such an assay results in a binary answer (i.e. either the RNA moiety will hybridise or not) which is a characteristic ideally suited for the analysis of bi-allelic loci, e.g. SNP-loci. However, such a strategy cannot be applied for STR-probes, as these DNA regions are characterised by multiple possible alleles that differ in length rather than solely in sequence. Indeed, the sensing part of such a probe cannot be positioned in the centre of the probe, but more towards a terminus. Therefore, some structural adaptations (e.g. an anchor region and the positioning of the RNA-base) to this probe are indispensable. As the loci of interest are longer than SNP-loci, the destabilizing effect of a mismatch decreases. This implicates that the RNA-moiety will hybridize even when a mismatch occurs, complicating the method of assessment and data analysis.

Taken together, there is clearly still a high need to design an STR genotyping probe and method that results in a high signal-to-noise, has no design limitations and can be implemented in a portable device.

The present invention relates to a composition comprising:.

The present invention further relates to a composition comprising an array of oligonucleotide probes as described above wherein said quencher is attached to the <NUM>' or <NUM>' terminus of each of said probes.

The present invention further relates to a composition comprising an array of oligonucleotide probes as described above wherein the said fluorophore is attached to a nucleotide of the second flanking region of each of said probes and wherein the said quencher is attached to a nucleotide of the specific DNA sequence of interest of each of said probes.

In a specific embodiment of this invention, the said fluorophore is a fluorescein derivate.

In a specific embodiment of this invention, the said quencher is an Iowa Black FQ quencher.

The present invention further relates to a composition comprising an array of oligonucleotide probes as described above containing more than one ribonucleotide.

The present invention further relates to a composition comprising an array of oligonucleotide probes as described above wherein said nucleotides are nucleic acid analogues.

The present invention further relates to a composition comprising an array of oligonucleotide probes as described above wherein each of said probes is immobilised on a support.

The present invention also relates to a method to genotype short tandem repeats within a sample comprising the steps of:.

The present invention further relates to a method to genotype as described above wherein said amplification within said sample is undertaken by an asymmetric PCR in order to obtain amplified, single stranded DNA sequences.

The present invention further relates to a method to genotype as described above wherein said amplification within said sample is undertaken by a symmetric PCR using biotin-labelled primers or a subsequent lambda exonuclease digestion in order to obtain amplified, single stranded DNA sequences.

The present invention further relates to a method to genotype as described above wherein said array of probes is added in solution, or is immobilised onto a support.

The present invention relates to a composition comprising an array of oligonucleotide probes and the RNase H2 enzyme. A probe is herein defined as a synthetically manufactured oligonucleotide, consisting of <NUM> or more nucleotides and/or ribonucleotides covalently linked to each other, wherein some nucleotides and/or ribonucleotides might be modified. Such a modification is defined as a molecule attached to the oligonucleotide that not necessarily occurs in natural DNA or RNA. Examples of modifications are e.g. the presence of a fluorescent moiety, the presence of a quencher, the presence of molecules for attachment purposes, modifiers of the melting temperature etc. Probes can be synthetically manufactured, but the definition of an oligonucleotide probe is herein not narrowed down to exclusively synthetically manufactured oligonucleotides. Probes are generally designed in such a way that they will interact with the investigated molecule, and the response of the probe upon this interaction will be observed and used in order to obtain information of the said investigated molecule.

DNA complementarity can be explained by Chargaff's rules, stating that adenine always forms hydrogen bonds with thymine or uracil, and cytosine with guanine, a process also referred to as Watson-Crick or Hoogsteen base pairing, resulting in double stranded DNA. Hybridisation or annealing is defined herein as the formation of a duplex or hetero-duplex structure, consisting of two nucleic acid strands after complementary base pairing. A duplex structure is defined as a complex of <NUM> fully complementary base paired nucleic strands. A hetero-duplex structure is defined as a complex of <NUM> partially complementary nucleic acid strands, e.g. <NUM> DNA strands deferring by one or more <NUM>-nucleotide repeats.

The function of the array of probes disclosed by this invention is genotyping of Short Tandem Repeat-loci (STR-loci). STR-loci are characterised by short (typically <NUM> nucleotides) repeating sequences that are polymorphous within a certain population regarding the amount of repeats. In contrast to Single Nucleotide Polymorphisms (SNPs), these loci are multi-allelic, indicating that a rather broad range of repeat numbers occur within the population. By determining the repeat number of sufficient loci, a statistically unique profile is obtained for an individual. The wording 'array of probes' refers to the fact that for each allele of the investigated STR-locus, a dedicated probe is designed. The array of probes consists of all different probes for a certain locus. The interaction between a specific probe and a sample should be analysed separately, implicating that all different probes should be physically separated, for example by means of different wells on a multi-well plate, or by immobilizing them in distinct spots on a surface.

The oligonucleotide probes disclosed by this invention comprise, from <NUM>' to <NUM>' or from <NUM>' to <NUM>' of a first flanking region, a specific DNA sequence of interest and a second flanking region, as illustrated in <FIG>.

The first flanking region is a sequence of nucleotides and comprises at least one nucleotide. In a more convenient embodiment of this invention, the flanking region comprises between <NUM> and <NUM> nucleotides. The first flanking region is complementary to and anneals with the region directly next to the STR-region and ensures proper annealing of the sample and the probe, therefore acting as an anchor. As this first flanking region has a pronounced higher melting temperature as compared to the second flanking region, initiation of the hybridisation is privileged at the first flanking region. In order to obtain correct genotyping, it is crucial that the first repeat of the sample anneals to the first repeat of the probe, and slippage of the sample is prevented.

The specific DNA sequence of interest comprises at least <NUM> short tandem repeat and contains at least one fluorophore and anneals with the short tandem repeat region within the sample. In one embodiment of this invention, this sample is DNA where the target STR-regions are amplified by means of e.g. polymerase chain reaction.

The second flanking region comprises at least <NUM> nucleotide and contains at least one ribonucleotide, e.g. ATP, CTP, GTP and UTP and contains at least one quencher moiety capable of efficiently quenching the said fluorophore.

Fluorophores are herein defined as compounds characterised with a fluorescent emission maximum between about <NUM> and <NUM>. A commonly used fluorescein derivate is <NUM>-FAM (<NUM>-carboxyfluorescein). Other commonly used fluorophores are <NUM>-Hexachloro-Fluorescein, <NUM>-Hexachloro-Fluorescein, <NUM>-Tetrachloro-Fluorescein5-TAMRA (<NUM>-carboxytetramethylrhodamine), <NUM>-TAMRA (<NUM>-carboxytetramethylrhodamine), Cy5 (Indodicarbocyanine-<NUM>); Cy3 (Indo-dicarbocyanine-<NUM>), and BODIPY FL (<NUM>,<NUM>-dibromo-<NUM>,<NUM>-difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-proprionic acid).

A quencher is defined as a moiety that suppresses luminescence of a fluorophore moiety when brought into proximity of said fluorophore. A common mechanism of quenching is fluorescence resonance energy transfer (FRET), but the definition of a quencher is herein not narrowed down to this mechanism. Other mechanisms are e.g. photo-induced electron transfer. Commercially available quenchers are: Dabcyl, Iowa Black® FQ and RQ, ZEN™ and Black Hole quenchers, e.g. BHQ-<NUM>®.

In a specific embodiment of this invention, a fluorescein derivate is used in combination with an Iowa Black FQ quencher moiety. Those skilled in the art will recognise 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 possible combination of fluorophore and quencher is Cy3 with Black Hole Quencher <NUM>.

The fluorophore and the quencher moiety are separated from each other by at least one ribonucleotide. If the quencher and the fluorescent moiety were to be linked to the same nucleotide or ribonucleotide, no signal would occur upon digestion of the probe by a suitable enzyme, as both said moieties would not be separated from each other. In a more specific embodiment of this invention, the fluorophore and the quencher are separated by <NUM> to <NUM> nucleotides.

In a specific embodiment of this invention, the quencher is attached to the <NUM>' or <NUM>' terminus of the probe.

The present invention further relates to oligonucleotide probes as described above wherein the said fluorophore is attached to a nucleotide of the second flanking region and wherein the said quencher is attached to a nucleotide of the specific DNA sequence of interest. The present invention further relates to oligonucleotide probes as described above which comprises more than one ribonucleotide.

The present invention further relates to oligonucleotide probes as described above wherein said nucleotides are nucleic acid analogues, e.g. LNA, PNA, GNA, TNA, morpholino (PMO).

The said probes described in this invention are functional both in solution and immobilised on a support.

After activation of the RNase enzyme at a high temperature, the mixture is cooled down slowly. In a specific embodiment of this invention, the mixture is cooled down at a rate of <NUM> per minute. However, it should be noted that both faster and slower cooling is feasible. Upon cooling down of the mixture, the probes will hybridise with the amplified DNA strands of the sample. Owing to the presence of an anchor region in the probe, hybridisation is privileged at the anchor-side of the probe. This ensures that the first repeat of the probe, starting from the anchor-side, will hybridise to the first repeat of the sample.

If said probe and the amplified DNA-strand have the exact same number of repeats, the complete probe will hybridise to the sample. When the probe and the amplified DNA-strand do not share the same repeat number, hetero-duplex formation will occur (<FIG>). In the latter situation, hybridisation will occur at a lower temperature as compared to the situation of full complementarity. As the RNase H2 enzyme is active in a broad range of temperatures, the probe is cleaved as soon as it hybridises to the sample (<FIG>). As a consequence, the fluorescent signal of probes with the same number of repeats as the sample increases at a higher temperature as compared to mismatch probes (<FIG>).

The present invention thus describes an STR-assay determining the hybridisation temperature of a probe by enzymatic digestion. The destabilizing effect of a partial mismatch between probe and sample is hard to assess for STR-loci, as these are by definition long loci. It is generally known that the longer a probe is, the lower the destabilizing effect of a mismatch is. By introducing an enzymatic cleavage step that relies on the specific hybridisation of an RNA unit in the probe, extremely sharp and distinct peaks are obtained, thereby optimally highlighting the difference in duplex stability. Only after specific hybridisation of this ribonucleotide, implying the formation of an open loop structure in the hetero-duplex (as illustrated in <FIG>), a signal is generated. The use of a fluorescent molecule in combination with a quencher moiety results in a high signal-to-noise ratio.

The present invention further relates to a method to genotype as described above wherein said amplification within said sample is undertaken by an asymmetric PCR in order to obtain amplified, single stranded DNA sequences. Obtaining single stranded DNA is crucial, as re-annealing of double stranded amplicons would be favoured above probe hybridisation. Asymmetric PCR is an often used technique to obtain single stranded DNA. In order to obtain this goal, the primers are added to the PCR reaction mixture in a different concentration. The primer that will be incorporated in the strand complementary to the probe will be added in excess. During the first PCR cycles, both primers will be consumed and PCR will occur exponentially. Upon depletion of the primer added in a lower concentration, PCR will occur linearly, as only the desired strand is produced.

An alternative for asymmetric PCR is symmetric PCR using a biotin-labelled primer. After PCR, the streptavidine beads are added to the amplified DNA. The biotin labelled primers will react covalently with the streptavidine, upon denaturation of the double stranded amplicons, the desired strand can be isolated. Another alternative is symmetric PCR with subsequent lambda exonuclease digestion. Only strands originating from a <NUM>' phosphate labelled primer will be digested.

The present invention also relates to a method as described above wherein said probes are added in solution, or are immobilised onto a support.

<NUM> different probes (having <NUM>, <NUM> and <NUM> repeats) designed for the TH01 locus were mixed with a synthetically manufactured complement that has <NUM> repeats. Concentration of the probes was <NUM>, concentration of the synthetic complement was <NUM>. Probe sequences can be found in table <NUM>. After adding RNase H2 enzyme, the mixture was heated to <NUM> for <NUM> minutes. Hereafter, the mixture was slowly cooled down in order to ensure proper hybridisation of the probes and the synthetic complement. During this hybridisation phase, fluorescence was monitored. The first derivative of the fluorescence with respect to the temperature was calculated.

Fluorescence dropped in all <NUM> wells, indicating that all different probes were digested by the enzyme. However, the matching probe was digested at a higher temperature as compared to the mismatch probes. This indicates hetero-duplex formation of the mismatch probes and the sample.

A buccal swab was immersed in a volume of <NUM>µL sterile HPLC-water. After a vortex-step of <NUM>", the swab was removed and the water was used as input for PCR. Singleplex asymmetric PCR was performed with <NUM>µL of input sample. Primer concentrations were <NUM> forward primer and <NUM> reverse primer. The volume of the PCR mixture was <NUM>µL, containing MgCl<NUM>+ at a concentration of <NUM>, dNTP's at <NUM> each, 1X Qiagen PCR buffer and <NUM> U HotStarTaq enzyme. Activation of the polymerase was done by heating the PCR mix at <NUM> for <NUM> minutes followed by <NUM> cycles of <NUM> for <NUM> minute, <NUM> for <NUM> minute and <NUM> for <NUM> seconds. Primer sequences can be found in table <NUM>.

After asymmetric PCR, aliquots of <NUM>µL amplified product were divided in a <NUM>-Well plate. To each separate well, <NUM>µL of one particular probe was added at a starting concentration of <NUM>. These mixtures were denatured for <NUM> minutes at <NUM>, followed by slowly cooling at a ramp rate of <NUM>/s while fluorescence was continually measured using a LightCycler (Roche). The first derivative of the hybridisation curve is calculated, resulting in melting peaks. Probe sequences can be found in table <NUM>. The sample was genotyped using CE-analysis and had alleles <NUM> and <NUM>.

The first derivative of all hybridisation curves is shown in <FIG>. A pronounced signal can be observed for alleles <NUM>, <NUM> and <NUM>. Although probe <NUM> is the longest probe of the <NUM> probes displaying a signal, it shows a distinctly lower hybridisation temperature, indicating hetero-duplex formation. The other probes show barely any signal, indicating that the mismatch was too destabilising for hybridisation to occur.

A buccal swab was prepared, amplified and analysed as described in example <NUM>. The same primers and probes were used as described in example <NUM>. The examined sample was genotyped using CE having allele <NUM>. Samples with allele <NUM> have <NUM> repeats but are characterised by a deletion of <NUM> nucleotide in their <NUM>rd repeat. These are challenging alleles as hybridisation of this sample with probe <NUM> results in a hetero-duplex only being destabilised by a one-nucleotide indel (insertion/deletion).

After asymmetric PCR, aliquots of <NUM>µL amplified product were divided in a <NUM>-Well plate. To each separate well, <NUM>µL of probe (<NUM>) was added. These mixtures were denatured for <NUM> minutes at <NUM>, followed by slowly cooling at a ramp rate of <NUM>/s while fluorescence was continually measured using a LightCycler (Roche). The first derivative of the hybridisation curve is calculated, resulting in melting peaks.

Claim 1:
A composition comprising:
a) an array of oligonucleotide probes wherein each of said probes comprises, from <NUM>' to <NUM>' or from <NUM>' to <NUM>, the following <NUM> regions:
I. a first flanking region comprising at least one nucleotide, which anneals with a region directly next to the specific DNA sequence of interest and which has a higher melting temperature than the second flanking region,
II. a region comprising a specific DNA sequence which anneals with the short tandem repeat region of interest within a sample and which contains at least one fluorophore, and
III. a second flanking region comprising at least <NUM> nucleotides, and which contains at least one ribonucleotide and at least one quencher moiety capable of efficiently quenching said fluorophore, wherein the fluorophore and the quencher moiety are separated from each other by at least one ribonucleotide, and
b) the RNase H2 enzyme capable of digesting said probe by recognition of the RNA:DNA duplex upon hybridization of said probe with said sample.