Melting temperature method, kit and reporter oligonucleotide for detecting mutant nucleic acids - Patents.com

Abstract

The present invention relates to a melting analysis-based method for detecting the presence of a variant sequence in a target nucleic acid sequence containing a nucleotide of interest, particularly for detecting microsatellite instability. The method uses a probe as a reporter oligonucleotide having a fluorophore and a quencher, and the nucleotide sequence contains a nucleotide with a hydrophobic intercalating residue. Also disclosed are methods for determining the effectiveness of a drug and for predicting the presence of a clinical disorder in an individual, as well as reporter oligonucleotides and kits for carrying out the methods.

Description

[Technical field]

[0001] The present invention relates to methods for detecting the presence of mutant sequences in target nucleic acid sequences containing a nucleotide of interest, in particular for detecting germline and somatic mutations and microsatellite instability. Methods for predicting the efficacy of drugs and for detecting the presence of clinical disorders in individuals, as well as reporter oligonucleotides and kits for carrying out the methods, are also disclosed. [Background technology]

[0002] Hereditary (germline) or non-hereditary (somatic) mutational events as single base substitutions, deletions, insertions, translocations and duplications have instrumental impact on human biology. Determination of the genetic profile reveals information on the response to environmental factors such as exercise and diet and on disease diagnosis, prognosis and applicable therapeutic regimens. Genetic evaluation is typically performed with tools such as DNA sequencing or allele-specific PCR. Methods involving variable binding of molecular probes are applicable not only to germline mutations but also to somatic mutations with low allele frequency when combined with techniques such as HRM.

[0003] Repetitive nucleotide sequences, such as direct or inverted repeats, are observed in many organisms. As an example, hundreds of thousands of microsatellite loci are distributed throughout the human genome, and thus statistically occur approximately once every 100,000 base pairs. Microsatellite loci are regions of genomic DNA that contain short tandem repeats, with the shortest repeat unit typically being 1-5 nucleotides in length. Thus, the repeat units of a particular microsatellite locus are commonly referred to as mono-, di-, tri-, tetra-, or pentanucleotide repeat loci, as applicable. A given microsatellite locus typically contains approximately 10-40 of these repeat units in a tandem arrangement. Furthermore, each microsatellite locus in normal genomic DNA of most diploid species, such as genomic DNA from mammalian species, contains two alleles at each locus. The two alleles may be identical or different in length from one another and may vary from individual to individual.

[0004] Microsatellite instability (MSI), or replication errors (RER), is one example of genomic instability that occurs in certain human neoplasms, where tumor cells have a reduced ability to accurately replicate their DNA. MSI is a common marker of underlying functional inactivation of human DNA mismatch repair (MMR) genes. Loss of function of MMR genes is thought to occur due to biallelic inactivation via coding region mutations, loss of heterozygosity (LOH), and / or promoter methylation. In addition, germline mutations in MMR genes are known to be autosomal dominant genetic defects in most hereditary nonpolyposis colon cancer (HNPCC) families. Other mutations induced by tumor cells in HNPCC individuals result in biallelic inactivation of certain MMR genes, causing loss of accurate replication of microsatellite DNA in tumors. MSI is therefore a marker of underlying DNA mismatch repair defects and is also associated with enhanced mutation rates of coding DNA. This mutator phenotype resulting from MMR deficiency causes both base substitutions in coding regions and frameshift mutations in direct repeats at equal frequency, in addition to resulting in MSI. The development of MMR deficiency and the resulting mutator phenotype are thought to be early events in tumorigenesis.

[0005] MSI is not only associated with germline defects in HNPCC families, but is also found in approximately 15-20% of sporadic colorectal cancers, a finding that also reflects an overall increase in genomic instability (also measured as tumor mutation burden). Findings of MSI deficiency in tumors are also associated with better prognosis in stage-matched tumors. Thus, identifying tumors with MSI is clinically important not only to imply germline MMR deficiency (HNPCC families), but also for prognostic stratification. Although clinical (Bethesda guidelines (Rodriguez-Bigas et al., 1997) and histopathological features may raise suspicion that a colorectal tumor is microsatellite unstable and possibly arises in HNPCC families, clinicopathological features are often insufficient to diagnose the presence of MSI. Thus, molecular testing may be utilized to elucidate the MSI status of clinically suspicious tumors (Boland et al., 1998).

[0006] In addition to colorectal tumors, MSI is also associated with other types of cancer and other genetic diseases.To explain, these include, among others, pancreatic cancer, gastric cancer, bladder cancer, prostate cancer, lung cancer, uterine cancer and breast cancer.Other representative gene sequences that are considered to be associated with microsatellite instability include, for example, Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), myotonic dystrophy (DM), fragile X syndrome, FRAXE mental retardation and spinocerebellar ataxia (SCA), Bruton's X-linked agammaglobulinemia (XLA), Bloom's syndrome (BS), craniofrontonasal syndrome (CFNS) and idiopathic pulmonary fibrosis (IPF).

[0007] In view of the above, it is apparent that analysis of variant nucleotide sequences, e.g., repetitive nucleotide sequences such as microsatellites, has many diagnostic and prognostic applications, among others. Thus, there is a need for sensitive, objective and reliable assays. Summary of the Invention

[0008] The present invention is as defined in the claims. The methods described herein represent rapid, easy, unbiased and sensitive methods for investigating germline and somatic mutations and microsatellite instability.

[0009] Provided herein is a method for detecting the presence of a variant sequence in a target nucleic acid sequence comprising a nucleotide(s) of interest (NOI) that consists of two strands and preferably contains repeats, said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said method comprising the following steps: a) providing a first sample containing nucleic acid suspected of containing the mutant sequence; b) preparing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence comprising the NOI; e) amplifying a target nucleic acid sequence in the presence of the first sample, the first primer, and the second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a variant sequence; and amplifying a target nucleic acid sequence in the presence of the second sample, the first primer, and the second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a melting analysis, such as a high resolution melting (HRM) analysis, of the first amplicon, thereby obtaining a first profile characterized by a first melting curve, and performing a melting analysis, such as a HRM analysis, of the second amplicon, thereby obtaining a second profile characterized by a second melting curve, the second profile being a reference profile characterized by a reference melting curve (each amplicon comprises a first strand and a second strand, and the melting analysis comprises hybridization of a reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by a fluorophore, and obtaining the first and second melting curves); (The reporter oligonucleotide is a sequence of 10 to 50 nucleotides, preferably 15 to 50 nucleotides, into which 2 to 10 hydrophobic nucleotides are inserted. the reporter oligonucleotide comprises, preferably at or within 4 nucleotides from its 5' end, a first fluorophore, and, preferably at or within 4 nucleotides from its 3' end, a first quencher; and The reporter oligonucleotide comprises a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; and the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence being free of repeats and capable of hybridizing to the first and second amplicons when the hybridization sequence hybridizes thereto; and g) comparing the first profile to a reference profile, wherein a difference between the first profile and the reference profile indicates that the first sample contains a variant sequence. Includes.

[0010] Provided herein is a method for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said method comprising the steps of: a) providing a first sample containing nucleic acid suspected of containing the mutant sequence; b) preparing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence; e) amplifying a target nucleic acid sequence in the presence of the first sample, the first primer, and the second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a mutant sequence; and amplifying a target nucleic acid sequence in the presence of the second sample, the first primer, and the second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a high resolution melting (HRM) analysis of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of the second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, the second HRM profile being a reference profile characterized by a reference melting curve, each amplicon comprising a first strand and a second strand, the HRM analysis comprising hybridization of a reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by a fluorophore, and obtaining the first and second melting curves; (Reporter oligonucleotides are sequences of 10-50 nucleotides with 2-10 hydrophobic nucleotides inserted therein. the reporter oligonucleotide comprises, preferably at or within 4 nucleotides from its 5' end, a first fluorophore, and, preferably at or within 4 nucleotides from its 3' end, a first quencher; and The reporter oligonucleotide comprises a hybridization sequence H, at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; and g) comparing the first HRM profile with a reference HRM profile, wherein a difference between the first HRM profile and the reference HRM profile indicates that the first sample contains a variant sequence. Includes.

[0011] Also provided herein is a kit-of-parts for detecting the presence of a variant sequence in a target nucleic acid sequence comprising a nucleotide(s) of interest (NOI) consisting of two strands and preferably comprising repeats, said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said kit-of-parts comprising: a) a reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end. (The reporter nucleotide is a sequence of 10 to 50 nucleotides, preferably 15 to 50 nucleotides, in which 2 to 10 hydrophobic nucleotides are inserted; and The reporter oligonucleotide comprises a hybridization sequence H, and the hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid; and the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence being free of repeats and capable of hybridizing to the first and second amplicons when the hybridization sequence hybridizes thereto; and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence; Includes.

[0012] Also provided herein is a kit-of-parts for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said kit-of-parts comprising: a) a reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end. (The reporter oligonucleotide is a sequence of 10-50 nucleotides having 2-10 hydrophobic nucleotides inserted therein, the reporter oligonucleotide including a hybridization sequence H), (at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid containing the reference sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid; and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence; Includes.

[0013] Also provided herein is a reporter oligonucleotide consisting of two strands, capable of hybridizing to one strand of a target nucleic acid sequence comprising a nucleotide(s) of interest (NOI), preferably comprising a repeat, said reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end, said reporter oligonucleotide being a sequence of 10-50 nucleotides, preferably 15-50 nucleotides, with 2-10 hydrophobic nucleotides inserted therein, said reporter oligonucleotide comprising a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid; The hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence being free of repeats and capable of hybridizing to the second strands of the first and second amplicons when the hybridization sequence hybridizes thereto.

[0014] Also provided herein is a reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end, wherein the reporter oligonucleotide is a sequence in the range of 10-50 nucleotides interspersed therewithin, in the range of 2-10 hydrophobic nucleotides, wherein the reporter oligonucleotide comprises a hybridization sequence H; at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and The hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid.

[0015] Also provided herein is a method for predicting the efficacy of a treatment for a clinical condition in an individual, comprising the steps of: a. Preparing a sample from said individual b. performing a method for detecting the presence of a variant sequence as described herein to determine whether the sample contains the variant sequence. wherein the presence of said variant sequence indicates whether said agent will be effective in treating said clinical condition in said individual.

[0016] Also provided herein are methods that are useful for predicting the presence, or even diagnosing, of any clinical condition associated with a particular mutation in an individual, comprising the steps of: a) preparing a sample from said individual; b) detecting the presence of a mutation associated with a clinical condition in the sample by carrying out a method for detecting a variant sequence as described herein. wherein the presence of said variant sequence is indicative of said individual suffering from said clinical condition. [Brief description of the drawings]

[0017] [Figure 1]Principle of the method for detecting variant nucleic acid sequences containing a NOI. (A) A target nucleic acid (black twisted line) is provided. The second strand contains the NOI (light grey). The first strand is complementary (black line). The arrows indicate the primers. The first primer hybridizes to the second strand, resulting in amplification of the primer (dashed line). The second primer hybridizes to the first strand, resulting in amplification of the primer (dashed line). For ease of overview, only the amplified part of the target nucleic acid is shown. Here, the second primer is provided in excess compared to the first primer (asymmetric PCR). Thus, more DNA containing the NOI sequence is generated (dashed black line and light grey dashed line). (B) The amplified DNA consists of a mixture of double-stranded target nucleic acid and single-stranded target nucleic acid containing the NOI. Here, the single-stranded sequence containing the NOI is shown (light grey). A reporter oligonucleotide (RO) hybridizes to that strand. It comprises here a fluorophore F' (circle) and a quencher Q (square). A melting curve is obtained (x-axis: T is temperature; y-axis: F is fluorescence). [Diagram 2] Microsatellite instability does not necessarily result in a significant change in melting temperature. BAT25 assay using asymmetric PCR. X-axis indicates temperature. (A) Melting curves to which bilinear normalization with an intensity threshold of 0.1 RFU and temperature shift was applied. The difference between the curves, measured as the maximum difference in amplitude between the curves, was approximately 0.07 RFU. (B) In the first negative derivative curve without the application of a temperature shift, the clean tissue has a Tm of 57.31°C and for the tumor tissue, the Tm is 57.41°C. [Diagram 3] Bilinear normalization makes the slope of the melting curve closer to zero before and after the actual melting phase. Microsatellite NR22 is analyzed using asymmetric PCR and a reporter oligonucleotide. The x-axis indicates temperature. HRM curves were obtained using: (A) standard normalization, no temperature shift; (B) bilinear normalization, no temperature shift. [Figure 4]Bilinear normalization makes the slope of the melting curve closer to 0 before and after the actual melting phase. Difference HRM curves from melting curves in FIG. 3, where the reference HRM curve is set as the baseline and it is subtracted from the other (here the first) melting curve. The X-axis represents temperature. (A) Bilinear normalization, no temperature shift; (B) Bilinear normalization, no temperature shift. [Diagram 5] Theoretically, there should be no difference in melting temperature between normal and tumor tissue samples from microsatellite stable individuals. However, differences in amounts, salt concentrations, impurities and other variables between samples can result in slight differences in melting temperature. However, bilinear normalization and temperature shift can reduce the difference between normal and tumor tissues of microsatellite stable patients. NR24 assay using asymmetric PCR and reporter oligonucleotides. X-axis indicates temperature. (A) HRM profile with standard normalization applied. The difference between the curves, measured as the maximum difference in fluorescence at a given temperature between normalized regions for the difference graph, was about 0.04 RFU. X-axis indicates temperature. (B) HRM profile with bilinear normalization applied but no temperature shift threshold applied. The difference between the curves was about 0.02 RFU. (C) HRM curve with bilinear normalization and temperature shift applied with an intensity threshold of 0.1 RFU. The difference between the curves was about 0.01 RFU. [Figure 6] Temperature shifts can counteract changes in melting temperatures caused by different salt concentrations in the DNA buffer. NR24 assay using asymmetric PCR and reporter oligonucleotides. The x-axis indicates temperature. (A) HRM curves where bilinear normalization was applied but no temperature shift was applied. The difference between the curves, measured as the maximum difference in fluorescence at a given temperature between normalized regions for the difference graph, was approximately 0.07 RFU. (B) HRM curves where bilinear normalization and a temperature shift intensity threshold of 0.1 RFU was applied. The difference between the curves was approximately 0.01 RFU. [Figure 7]Assay for the investigation of the microsatellite MONO27 using asymmetric PCR on 16 normal tissue DNA samples. HRM curves were obtained with: (A) bilinear normalization, no temperature shift; (B) bilinear normalization, and with a temperature shift at an intensity threshold of 0.1 RFU. The x-axis indicates temperature. The application of a temperature shift allows the use of one sample as a universal reference. [Figure 8] Difference plots of the HRM curves from Figure 7. (A) Bilinear normalization, no temperature shift; (B) Bilinear normalization, and temperature shift at an intensity threshold of 0.1 RFU. The x-axis indicates temperature. [Figure 9] Asymmetric PCR creates more single-stranded target amplicons. NR22 assay using symmetric and asymmetric PCR. The X-axis shows PCR cycle number and the Y-axis shows fluorescence. [Figure 10] Asymmetric PCR creates a higher signal-to-noise ratio and a sharper melt (i.e., narrower melt peak). NR22 assay using symmetric and asymmetric PCR. (A) Melting curves; (B) Normalized melting curves using standard normalization. The X-axis indicates temperature. [Figure 11] Asymmetric PCR facilitates the differentiation of MSS and MSI patients. Data using the NR22 assay are shown. HRM curves were obtained after applying standard normalization using asymmetric PCR (A) or using symmetric PCR (B). The X-axis indicates temperature. [Figure 12] Asymmetric PCR facilitates the differentiation of MSS and MSI patients. Data using the NR22 assay are shown. Difference plots from the HRM curves of FIG. 11. (A) Asymmetric PCR; (B) Symmetric PCR. The X-axis indicates temperature. [Figure 13] A single terminal overhang in the reporter nucleotide can increase the melting temperature. Data based on NR22 using asymmetric PCR using a reporter oligonucleotide with two different reporter oligonucleotides is shown. (A) HRM curve. (B) Negative first derivative of the HRM curve. The X-axis indicates temperature. [Figure 14]Double quenching the reporter oligonucleotide increases the signal to noise ratio. Data using the BAT26 assay using asymmetric PCR are shown. HRM curves (A) and negative first derivative curves (B) using single quenched ("no DQ probe") and double quenched ("DQ probe") reporter oligonucleotides. The X-axis indicates temperature. [Figure 15] Additional nucleotide repeats in the hybridization sequence of the reporter oligonucleotide allow for longer microsatellites to be detected. Data using the NR21 assay using asymmetric PCR is shown. The X-axis indicates temperature. [Figure 16] A single point mutation changes the melting temperature by several degrees. The KIT exon 13 assay was used for the experiments. PCR was performed as asymmetric PCR. The X-axis indicates temperature. The dashed line indicates the results for the wild type target nucleic acid, and the solid line indicates the results for the target nucleic acid containing the mutation. (A) Normalized HRM curves; the Y-axis indicates fluorescence. (B) Melting peaks (negative first derivative of the melting curve). The Y-axis is -dF / dT. [Figure 17] A strong helper sequence helps to distinguish between wild type and mutant. The NR24 assay was used for the experiments. PCR was performed as asymmetric PCR. The x-axis indicates temperature and the y-axis indicates fluorescence. The dashed line indicates the results for the wild type target nucleic acid and the solid line indicates the results for the target nucleic acid containing a mutation. (A) Normalized HRM curve using a first reporter oligonucleotide containing a first helper sequence. (B) Normalized HRM curve using a second reporter oligonucleotide containing a second helper sequence that is stronger than the first helper sequence. [Figure 18]A strong helper sequence aids in distinguishing between wild type and mutant. The NR24 assay was used for the experiments. PCR was performed as asymmetric PCR. The x-axis indicates temperature and the y-axis indicates fluorescence. The dashed line indicates the results for the wild type target nucleic acid and the solid line indicates the results for the target nucleic acid containing a mutation. (A) Difference plot using a first reporter oligo containing a first helper sequence. (B) Difference plot using a second reporter oligo containing a second helper sequence that is stronger than the first helper sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition Amplicon "Amplicon" refers to a molecule created by copying or transcribing another molecule. Exemplary processes that can produce an amplicon include transcription, cloning, and / or polymerase chain reaction (PCR) or another nucleic acid amplification technique (e.g., strand displacement PCR amplification (SDA), duplex PCR amplification, etc.). Typically, an amplicon is a copy of or complementary to a selected nucleic acid (e.g., a template or target nucleic acid).

[0019] Bilinear normalization. The term "bilinear normalization" is used herein to refer to a mathematical transformation applied to the curve(s) by scaling the fluorescence data of the difference curve to a normalized curve. This allows the comparison of the difference curves removing other factors that may affect the fluorescence signal (such as different signal strengths among different positions in the instrument, different transparency of the plastic and other factors that introduce variables in the fluorescence measurement). Bilinear normalization forces the curve(s) to have the same value (at the average value of the normalization region) in the selected normalization region and the course of the curve to be as horizontal (flat) as possible. There are several algorithms known in the art that can be applied to the curve(s) and transform the curve(s) into a curve(s) that allows the comparison of different melting curves. Normalization also reduces the effect of different optical and mechanical differences between wells.

[0020] Hydrophobic nucleotides The term "hydrophobic nucleotide" as used herein refers to a hydrophobic nucleotide as described in detail herein below in the "Hydrophobic Nucleotide" section. In particular, a hydrophobic nucleotide according to the present invention comprises an intercalator connected to a nucleotide / nucleotide analog / backbone monomer unit via a linker.

[0021] Melting Temperature The term "melting temperature" as used herein refers to the temperature in degrees Celsius at which 50% helical (hybridized) versus coiled (unhybridized) form exists. Melting temperature is also referred to as (T m Melting of nucleic acids and nucleic acid analogs refers to the thermal separation of the two strands of a double-stranded nucleic acid molecule.

[0022] Microsatellite The term "microsatellite", "microsatellite marker" or "microsatellite locus" refers to a region of genomic DNA that contains tandem nucleotide repeats. These repeats or "repeat units" are typically about 1 to about 7 base pairs in length. A microsatellite locus typically contains about 10 to 40 of these repeat units in a tandem arrangement. In addition to mononucleotide repeats, which are repeats of one nucleotide, other representative repeating nucleotide sequences include dinucleotide repeats, e.g., AT repeats and GC repeats, trinucleotide repeats, e.g., CGG repeats, CGC repeats, TAT repeats, ATT repeats, tetranucleotide repeats, pentanucleotide repeats and / or their complementary repeats.

[0023] Nucleotide Analogues The term "nucleotide analog" includes all nucleotide analogs that can be incorporated into a nucleic acid backbone and are capable of specific base pairing essentially like naturally occurring nucleotides. Nucleotide analogs according to the present invention include PNA, HNA, MNA, ANA, LNA, XNA, INA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, α-L-ribo-LNA, α-L-xylo-LNA, β-D-xylo-LNA, α-D-ribo-LNA, [3.2.1]-LNA, bicyclo-DNA, 6-amino-bicyclo-DNA, 5-epi-bicyclo-DNA, α-bicyclo-DNA, tricyclo-DNA, bicyclo[4.3.0]-DNA, bicyclo[3.2.1]-DNA, bicyclo[4.3.0]amide-DNA, β-D-ribopyranosyl-NA, α-L-lyxopyranosyl-NA, 2'-R 1 -RNA, 2'-OR 1 -RNA(R 1 is a substituent), nucleotide analogs selected from the group consisting of α-L-RNA, α-D-RNA, and β-D-RNA.

[0024] Nucleotide of interest The term "nucleotide(s) of interest" as used herein refers to a nucleotide(s) in a target nucleic acid sequence that may exist in two different variants. A "nucleotide(s) of interest" may also be referred to herein as a "NOI". Thus, a NOI may consist of a variant sequence or may consist of a reference sequence, also referred to herein as a "reference sequence". In some embodiments, a reference sequence may be a wild type sequence.

[0025] nucleotide The term "nucleotide" as used herein refers to naturally occurring nucleotides, such as naturally occurring ribonucleotides or deoxyribonucleotides, or naturally occurring derivatives of ribonucleotides or deoxyribonucleotides.Naturally occurring nucleotides include deoxyribonucleotides that contain one of the four nucleobases adenine (A), thymine (T), guanine (G) or cytosine (C), and ribonucleotides that contain one of the four nucleobases adenine (A), uracil (U), guanine (G) or cytosine (C).

[0026] Oligonucleotides As used herein, the term "oligonucleotide" refers to an oligomer of nucleotides and / or nucleotide analogs and / or hydrophobic nucleotides. Preferably, the oligonucleotide is an oligomer of nucleotides, optionally including one or more hydrophobic nucleotides.

[0027] Reference target sequence The term "reference target sequence" refers to a stretch of a target nucleic acid sequence that comprises a reference sequence.

[0028] standard normalization The term "standard normalization" refers to the scaling of the difference curves to a normalized curve of the fluorescence data, allowing for comparison of the difference curves that removes other factors that may affect the fluorescence signal (such as different signal strengths among different locations in the instrument, different transparency of plastics, and other factors that introduce variables in the fluorescence measurements).

[0029] Target Nucleic Acid Sequence The term "target nucleic acid sequence" as used herein refers to a nucleic acid sequence that includes a nucleotide(s) of interest (NOI). The target nucleic acid sequence can be amplified using a set of primers.

[0030] Temperature Shift The term "temperature shift" as used herein refers to two or more normalized curves that have exactly the same T at a given fluorescence limit (intensity threshold). m Applying such a transformation algorithm to a data set reduces effects from, for example, different salt concentrations in different samples.

[0031] Mutant sequences The term "mutant sequence" as used herein refers to a nucleotide(s) in a target nucleic acid sequence that is different from a reference sequence. Thus, a target nucleic acid sequence may contain a NOI that is a mutant sequence, or a target nucleic acid sequence may contain a NOI that is a reference sequence, or a NOI may be yet another mutant sequence. A mutant sequence may be a mutation, such as a single base mutation, or it may be an insertion or deletion. A mutant sequence and a reference sequence may overlap such that one sequence contains the entire sequence of the other. In such a case, a mutant sequence and a reference sequence may differ in the number of nucleotides that make up the sequence.

[0032] Methods for detecting the presence of a variant sequence in a target nucleic acid sequence The present invention relates to a method for detecting mutant sequences in a target nucleic acid sequence.Therefore, the method is particularly useful for detecting the presence of a specific sequence in a target nucleic acid sequence, which may occur in two or more different sequences.The method can be useful, for example, for distinguishing between wild-type and mutant sequences, and can be useful for distinguishing between different polymorphic sequences.The method is particularly useful for detecting mutant sequences resulting from microsatellite instability by detecting the presence of mutant sequences that have different lengths from wild-type or reference sequences.Therefore, the method can be advantageously used to detect target nucleic acid sequences that contain repeats, particularly microsatellites.

[0033] Provided herein is a method for detecting the presence of a variant sequence in a target nucleic acid sequence comprising a nucleotide(s) of interest (NOI) that consists of two strands and preferably contains repeats, said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said method comprising the following steps: a) providing a first sample containing nucleic acid suspected of containing the mutant sequence; b) preparing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence comprising the NOI; e) amplifying a target nucleic acid sequence in the presence of the first sample, the first primer, and the second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a mutant sequence; and amplifying a target nucleic acid sequence in the presence of the second sample, the first primer, and the second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a melting analysis, such as a high resolution melting (HRM) analysis, of the first amplicon, thereby obtaining a first profile characterized by a first melting curve, and performing a melting analysis, such as a HRM analysis, of the second amplicon, thereby obtaining a second profile characterized by a second melting curve, the second profile being a reference profile characterized by a reference melting curve, where each amplicon comprises a first strand and a second strand, and the melting analysis comprises hybridization of a reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by a fluorophore, and obtaining the first and second melting curves; (The reporter oligonucleotide is a sequence of 10 to 50 nucleotides, preferably 15 to 50 nucleotides, into which 2 to 10 hydrophobic nucleotides are inserted. the reporter oligonucleotide comprises, preferably at or within 4 nucleotides from its 5' end, a first fluorophore, and, preferably at or within 4 nucleotides from its 3' end, a first quencher; and The reporter oligonucleotide comprises a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; and the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence being free of repeats and capable of hybridizing to the first and second amplicons when the hybridization sequence hybridizes thereto; and g) comparing the first profile to a reference profile, wherein a difference between the first profile and the reference profile indicates that the first sample contains a variant sequence. Includes.

[0034] Provided herein is a method for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said method comprising the steps of: a) providing a first sample containing nucleic acid suspected of containing the mutant sequence; b) preparing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence; e) amplifying a target nucleic acid sequence in the presence of the first sample, the first primer, and the second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a mutant sequence; and amplifying a target nucleic acid sequence in the presence of the second sample, the first primer, and the second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a high resolution melting (HRM) analysis of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of the second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, the second HRM profile being a reference profile characterized by a reference melting curve, each amplicon comprising a first strand and a second strand, the HRM analysis comprising hybridization of a reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by a fluorophore, and obtaining the first and second melting curves; (Reporter oligonucleotides are sequences of 10-50 nucleotides with 2-10 hydrophobic nucleotides inserted therein. the reporter oligonucleotide comprises, preferably at or within 4 nucleotides from its 5' end, a first fluorophore, and, preferably at or within 4 nucleotides from its 3' end, a first quencher; and The reporter oligonucleotide comprises a hybridization sequence H, at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; and g) comparing the first HRM profile with a reference HRM profile, wherein a difference between the first HRM profile and the reference HRM profile indicates that the first sample contains a variant sequence. Includes.

[0035] Thus, when there are two or more different target nucleic acid sequences, the method is useful for distinguishing at least two different target nucleic acid sequences, namely, the target nucleic acid sequence that includes a variant sequence and the target nucleic acid sequence that does not include a variant sequence. The latter may also be called "reference sequence". Optionally, the method may be useful for distinguishing between target nucleic acid sequences that include other variant sequences. The general principle of the method is illustrated in FIG. 1. In short, the difference between the first and second profiles, in particular the difference between the analyzed melting profile and the reference melting profile, suggests that the tested sample (first sample) includes a variant sequence; it will be understood throughout this disclosure that the difference preferably refers to a significant difference.

[0036] In the embodiment of the present invention, the method is used to distinguish between wild-type and mutant sequences, the mutant sequence can be the reference sequence. However, in many cases, the wild-type sequence is the reference sequence. The method of the present invention can also be used to distinguish between wild-type and some different mutant sequences, in which case the wild-type sequence is typically the reference sequence and some different mutant sequences are the mutant sequences. In some embodiments of the method, the reference sequence is a microsatellite sequence. In that case, the mutant sequence can be a microsatellite sequence consisting of some tandem repeats, with a number of tandem repeats that differs from the number of tandem repeats of the reference sequence. For example, the mutant sequence can be the result of microsatellite instability - in these cases, the mutant sequence can be a plurality of mutant sequences with different numbers of tandem repeats. By comparison, the reference sequence then has a certain number of tandem repeats.

[0037] The mutant sequence may, in some embodiments, be a mutation indicative of a disease state, or it may be predictive of the efficacy of a given treatment.

[0038] Nucleotide(s) of interest (NOI), variant sequence and reference sequence A mutant sequence can be any sequence that differs from another sequence, particularly a reference sequence. In many cases, a mutant sequence is a variant sequence, which is a sequence that differs from a wild-type sequence, for example, due to the presence of a mutation that replaces a nucleotide with another, and / or due to the insertion and / or deletion of a nucleotide compared to a reference sequence. However, a mutant sequence can also be a polymorphic sequence or any other sequence that differs from a reference sequence. Preferably, the mutant sequence is a variant of a microsatellite, which has a length that differs from the normal length of the microsatellite. The present methods are in fact particularly useful for detecting microsatellite instability. They can be used to detect the instability of short (less than 15 nucleotides) microsatellites and longer microsatellites (more than 15 nucleotides). In subjects with microsatellite instability, the longer microsatellites typically mutate before the shorter microsatellites - therefore, it may be advantageous to use target nucleic acids that are longer microsatellites with a length of 15 nucleotides or more.

[0039] The variant sequence may consist of at least one, such as 1, such as 2, 3, such as 4, 5, such as 6, 7, such as 8, 9, such as 10, such as 10-20, such as 20-50, such as more than 50 nucleotides. Preferably, the variant sequence consists of 10 or more, such as 15 or more nucleotides. Thus, in some embodiments, the variant sequence consists of 11, 12, 13, 14, 15, 16, 17, 19, 20 or more nucleotides, such as 25, 30, 35, 40, 45 or 50 or more nucleotides. For example, the variant sequence consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides.

[0040] In some embodiments of the invention, the variant sequence is a single nucleotide mutation or single nucleotide polymorphism (SNP).

[0041] A mutant sequence may be a change in one or more nucleotides relative to one or more other nucleotides compared to a reference target sequence. Additionally, the term mutant sequence may be a deletion or insertion of a nucleotide in a nucleic acid, for example, a deletion or insertion of a nucleotide compared to a reference target sequence.

[0042] The target nucleic acid sequence may contain a polymorphic site (see details herein below), and thus the reference target sequence may contain one polymorphism, whereas the "variant sequence" may constitute another polymorphism.

[0043] In one embodiment, the reference target sequence is a wild type sequence, i.e. the sequence that occurs most frequently in nature, whereas the mutant sequence comprises one or more mutations, insertions or deletions compared to said wild type sequence. Thus, the mutant sequence according to the present invention may in one embodiment be a polymorphism, such as a single nucleotide polymorphism (SNP). For example, the polymorphism may indicate a specific DNA profile. Knowledge of a specific DNA profile can be used, for example, to identify an individual. For example, a specific DNA profile can be used to identify a crime or a potential crime, or to identify a corpse or a part of a corpse. Furthermore, a specific DNA profile can be used to determine relationships between individuals, for example, parent-child relationships or more distant relationships. Relationships can also be relationships between different species and different populations of a given species.

[0044] In some embodiments, the reference sequence is or includes a microsatellite having a given number of repeats, and the variant sequence can then be one or several variant sequences having a different number of repeats than that observed in the reference or wild type sequence.

[0045] In one embodiment, the variant sequence may be indicative of a clinical condition or the mutation may be indicative of an increased risk of a clinical condition. In particular, the variant sequence may be a microsatellite and the clinical condition may be associated with microsatellite instability.

[0046] The clinical condition can be selected from the group consisting of, for example, neoplastic diseases, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases including diabetes.

[0047] Furthermore, the variant sequence may suggest a specific response to a given drug treatment. For example, the presence of the variant sequence may suggest whether an individual will respond positively to the drug treatment or whether the individual can tolerate a specific drug treatment or not. For example, the detection of MSI can be used in a decision-making process to determine the use of checkpoint inhibitors such as, for example, Keytruda (registered trademark) (pembrolizumab, Merck and Company) or Opdivo (nivolumab, Bristol-Myers Squibb).

[0048] The variant sequence may be located in a specific gene, gene segment, microsatellite or any other DNA sequence. Furthermore, the variant sequence may be located in mRNA, miRNA or any other RNA sequence. The methods described herein enable the detection of specific DNA, which may be of eukaryotic, prokaryotic, archaeal or viral origin. For example, the present invention can assist in the diagnosis and / or genotyping of various infectious diseases by assaying for specific sequences known to be associated with specific microorganisms.

[0049] Target nucleic acid sequence The method is for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and containing the nucleotide(s) of interest (NOI). While the target nucleic acid sequence consists of two strands and is typically DNA, - for example, if the amplification is of RNA, it will be understood that the method of the present invention can be readily adapted to detect the presence of a variant sequence in the target nucleic acid by amplifying a single strand of the target nucleic acid sequence. Thus, the method can also be used, for example, to detect a variant sequence in a target RNA resulting from the transcription of a target nucleic acid sequence consisting of two strands.

[0050] The target nucleic acid is thus a sequence of interest, suspected of containing a mutant sequence, in other words it often corresponds to a particular genetic locus.

[0051] The target nucleic acid sequence may consist of at least one, such as 1, such as 2, 3, such as 4, 5, such as 6, 7, such as 8, 9, such as 10, such as 10-20, such as 20-50, more than 50 nucleotides. Preferably, the target nucleic acid sequence consists of 15 or more nucleotides. Thus, in some embodiments, the target nucleic acid sequence consists of 11, 12, 13, 14, 15, 16, 17, 19, 20 or more nucleotides, such as 25, 30, 35, 40, 45 or 50 or more nucleotides. For example, the target nucleic acid sequence consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. Since the method is particularly useful for detecting variant sequences of microsatellites, especially microsatellites having a length of 15 or more nucleotides, where the target nucleic acid sequence is a reference sequence, the length is preferably 15 or more nucleotides.

[0052] In some embodiments, the target nucleic acid is a microsatellite that contains many tandem repeats. For the reference sequence, the number of tandem repeats is M, where M is an integer. For the reference sequence, the total length is n nucleotides. The variant sequence has M' tandem repeats and a total length of n' nucleotides. M and M' are different integers, where n and n' are different integers. However, some variant sequences may have M tandem repeats and a total length of n nucleotides - but in this case, some or most variant sequences still have a different number of repeats and a different total length compared to the reference sequence, which allows the method to detect the presence of variant sequences that differ from the reference sequence.

[0053] In some embodiments, n and / or n' are 1, 1, such as 2, 3, such as 4, 5, such as 6, 7, such as 8, 9, such as 10, such as 10-20, such as 20-50, more than 50, etc. nucleotides. Preferably, n and / or n' are 15 or more nucleotides; in some embodiments, at least n is 15 or more nucleotides. Thus, in some embodiments, n and / or n' are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides, 25, 30, 35, 40, 45 or 50 or more nucleotides. For example, n and / or n' are 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. Preferably, n consists of at least 15, 16, 17, 18, 19, 20 or more nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides, such as 25, 30, 35, 40, 45 or 50 or more nucleotides. In some embodiments, n' is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides, such as 25, 30, 35, 40, 45 or 50 or more nucleotides.

[0054] The method can be used to detect mutant sequences that may be indicative of a particular disorder, for example a disorder associated with microsatellite instability.

[0055] It is often advantageous to examine a sample for the presence of several different variant sequences. Thus, the method may be adapted to detect several variant sequences simultaneously. Sometimes, it is sufficient to determine whether at least one of many variant sequences is present.

[0056] In some embodiments, the target nucleic acid sequence is a plurality of target nucleic acid sequences including one or more of BAT25, BAT26, NR21, NR22, NR24 and MONO27, which are microsatellite markers that contain mononucleotide repeats. The method allows for the detection of variant sequences that have a different number of repeats and therefore a different overall length from the reference sequences for these target nucleic acid sequences.

[0057] The reference or wild type sequence of human BAT25 is as shown in SEQ ID NO: 1. The mononucleotide repeats of BAT25 correspond to positions 147 to 171 of SEQ ID NO: 1. The reference sequence therefore has M mononucleotide repeats, where M=25.

[0058] The reference or wild type sequence of human BAT26 is as shown in SEQ ID NO: 2. The mononucleotide repeats of BAT26 correspond to positions 222 to 248 of SEQ ID NO: 2. The reference sequence therefore has M mononucleotide repeats, where M=27.

[0059] The reference or wild type sequence of human NR21 is as shown in SEQ ID NO: 3. The mononucleotide repeats of NR21 correspond to positions 189 to 209 of SEQ ID NO: 3. The reference sequence therefore has M mononucleotide repeats, where M=21.

[0060] The reference or wild type sequence of human NR22 is as shown in SEQ ID NO: 4. The mononucleotide repeat of NR22 corresponds to positions 152 to 172 of SEQ ID NO: 4. The reference sequence therefore has M mononucleotide repeats, where M=21.

[0061] The reference or wild type sequence of human NR24 is as shown in SEQ ID NO: 5. The mononucleotide repeats of NR24 correspond to positions 165 to 187 of SEQ ID NO: 5. The reference sequence therefore has M mononucleotide repeats, where M=23.

[0062] The reference or wild type sequence of human MONO27 is as shown in SEQ ID NO: 6. The mononucleotide repeats of MONO27 correspond to positions 300 to 327 of SEQ ID NO: 6. The reference sequence therefore has M mononucleotide repeats, where M=28.

[0063] In some embodiments, the target nucleic acid sequence is BAT25 as shown in SEQ ID NO:1, and the NOI corresponds to the sequence defined by positions 147 to 171 of SEQ ID NO:1. In other embodiments, the target nucleic acid sequence is BAT26 as shown in SEQ ID NO:2, and the NOI corresponds to the sequence defined by positions 222 to 248 of SEQ ID NO:2. In other embodiments, the target nucleic acid sequence is NR21 as shown in SEQ ID NO:3, and the NOI corresponds to the sequence defined by positions 189 to 209 of SEQ ID NO:3. In other embodiments, the target nucleic acid sequence is NR22 as shown in SEQ ID NO:4, and the NOI corresponds to the sequence defined by positions 152 to 172 of SEQ ID NO:4. In other embodiments, the target nucleic acid sequence is NR24 as shown in SEQ ID NO:5, and the NOI corresponds to the sequence defined by positions 165 to 187 of SEQ ID NO:5. In other embodiments, the target nucleic acid sequence is MONO27 as shown in SEQ ID NO:6, and the NOI corresponds to the sequence defined by positions 300 to 327 of SEQ ID NO:6.

[0064] In another embodiment, the target nucleic acid sequence is two target nucleic acid sequences, for example, the two target nucleic acid sequences are BAT25 and BAT26; BAT25 and NR21; BAT25 and NR22; BAT25 and NR24; BAT25 and MONO27; BAT26 and NR21; BAT26 and NR22; BAT26 and NR24; BAT26 and MONO27; NR21 and NR22; NR21 and NR24; NR21 and MONO27; NR22 and NR24; NR22 and MONO27; NR24 and MONO27, and the NOI is as defined above.

[0065] In another embodiment, the target nucleic acid sequence is three target nucleic acid sequences. For example, the three target nucleic acid sequences are BAT25, BAT26 and NR21; BAT25, BAT26 and NR22; BAT25, BAT26 and NR24; BAT25, BAT26 and MONO27; BAT25, NR21 and NR22; BAT25, NR21 and NR24; BAT25, NR21 and MONO27; BAT25, NR22 and NR24; BAT25, NR22 and MONO27; BAT25, NR24 and MONO27; BAT25, NR24 and MONO27; AT26, NR21 and NR22; BAT26, NR21 and NR24; BAT26, NR21 and MONO27; BAT26, NR22 and NR24; BAT26, NR22 and MONO27; BAT26, NR24 and MONO27; NR21, NR22 and NR24; NR21, NR22 and MONO27; NR21, NR24 and MONO27; NR22, NR24 and MONO27, where the NOIs are as defined above.

[0066] In another embodiment, the target nucleic acid sequences are four target nucleic acid sequences, for example, the four target nucleic acid sequences are BAT25, BAT26, NR21 and NR22; BAT25, BAT26, NR21 and NR24; BAT25, BAT26, NR21 and MONO27; BAT25, BAT26, NR22 and NR24; BAT25, BAT26, NR22 and MONO27; BAT26, NR21, NR22 and NR24; BAT26, NR21, NR22 and MONO27; BAT26, NR21, NR24 and MONO27; NR21, NR22, NR24 and MONO27, and the NOI is as defined above.

[0067] In another embodiment, the target nucleic acid sequences are five target nucleic acid sequences, for example, the five target nucleic acid sequences are BAT25, BAT26, NR21, NR22 and NR24; BAT25, BAT26, NR21, NR22 and MONO27; BAT25, BAT26, NR22, NR24 and MONO27; BAT26, NR21, NR22, NR24 and MONO27, and the NOIs are as defined above.

[0068] In another embodiment, the target nucleic acid sequences are six target nucleic acid sequences, for example the six target nucleic acid sequences are BAT25, BAT26, NR21, NR22, NR24 and MONO27, and the NOIs are as defined above.

[0069] In a preferred embodiment, the target nucleic acids are five target nucleic acids, preferably BAT25, BAT26, NR21, NR22 and NR24; or BAT25, BAT26, NR22, NR24 and MONO27, and the NOIs are as defined above. In another preferred embodiment, the target nucleic acid sequences are six target nucleic acid sequences, preferably BAT25, BAT26, NR21, NR22, NR24 and MONO27, and the NOIs are as defined above.

[0070] sample In the first step of the method, a first sample is provided, which comprises the nucleic acid suspected to comprise the mutant sequence to be detected.In the second step of the method, a second sample is provided, which comprises the reference sequence.The second sample is therefore the reference sample, and these terms are used interchangeably herein.

[0071] The sample may include a cell that contains said nucleic acid. The cell may be, for example, a prokaryotic or eukaryotic cell, such as a plant cell or a mammalian cell.

[0072] The sample can be, for example, a synthetically prepared sample, which may or may not be further processed in vitro, but most often the sample is a sample obtained from an individual.

[0073] In some embodiments, the first sample is a sample obtained from an individual who is suffering from or suspected of suffering from a disease or disorder characterized by the presence of variant sequence, and the second sample is a sample obtained from a healthy individual.For example, it is also possible to use a first sample that comprises cells from diseased tissue, i.e., cells suspected of containing variant sequence characteristic of disease from one individual who is suspected of suffering from disease, and a second sample that comprises cells from healthy tissue, i.e., cells from the same individual that do not contain variant sequence.This method, in some embodiments, allows the use of a universal reference sample.

[0074] Therefore, it is often desirable to test DNA or RNA of an individual, such as a mammal, for example a human. In such a case, the sample is a sample from said individual. Thus, the sample may comprise, for example, a nucleic acid selected from the group consisting of DNA, mRNA, miRNA or any other RNA sequence. The sample may be derived from a body fluid sample, such as a blood sample, a biopsy, a hair, a nail, or any other suitable sample. In an embodiment of the present invention in which an individual suffers from cancer, the sample may be a sample of a cancer tumor removed from the individual by surgery, or a biopsy of said tumor. However, in an embodiment of the present invention in which an individual suffers from cancer, the sample may also be a blood sample, which may typically comprise CTCs and cfDNA.

[0075] The sample may be processed in vitro prior to detection of the presence of the mutant sequence. For example, the sample may be subjected to one or more purification steps that can completely or partially purify nucleic acid from the sample. Furthermore, the sample may be subjected to reverse transcription.

[0076] Samples may contain complex biological mixtures of nucleic acids (RNA and DNA) and non-nucleic acids, such as intact cells or crude cell extracts.

[0077] If the target DNA is double stranded or otherwise has secondary and / or tertiary structures that may interfere with its detection, it may be necessary to heat it before carrying out the methods of the invention. In some cases, it may also be desirable to extract nucleic acids from a complex biological sample before carrying out amplification by any method known in the art.

[0078] The sample may include a wide range of eukaryotic and prokaryotic cells, including protoplasts; or other biological materials that may carry the target deoxyribonucleic acid. The method is therefore applicable to tissue culture animal cells, animal cells (e.g., blood, serum, plasma, reticulocytes, lymphocytes, urine, bone marrow tissue, cerebrospinal fluid, or any product prepared from blood or lymph) or any kind of tissue biopsy (e.g., muscle biopsy, liver biopsy, kidney biopsy, bladder biopsy, bone biopsy, cartilage biopsy, skin biopsy, pancreatic biopsy, intestinal biopsy, thymus biopsy, mammalian biopsy, uterine biopsy, testicular biopsy, eye biopsy, or brain biopsy homogenized in lysis buffer), plant cells or other cells that are sensitive to osmotic shock, as well as bacteria, yeast, viruses, mycoplasma, protozoa, rickettsia, fungal cells, and other small microbial cells, etc. The assay and isolation procedure of the present invention is useful, for example, for detecting non-pathogenic or pathogenic microorganisms of interest. By detecting the presence of the variant sequence in a biological sample, the presence of the microorganism can be established.

[0079] In some embodiments, at least the first sample is obtained from an individual suffering from or suspected of suffering from a disease or disorder characterized by the presence of a variant sequence, particularly the presence of a variant microsatellite sequence or the presence of a variant sequence, as detailed above. In some embodiments, the disease is cancer or a genetic disorder, such as pancreatic cancer, gastric cancer, bladder cancer, prostate cancer, lung cancer, uterine cancer, breast cancer, hereditary nonpolyposis colorectal cancer. In some embodiments, the disease is a genetic disorder, such as Lynch syndrome, Huntington's disease (HD), dentatorubral-pallidoluysian rhombocytopenia (DRPLA), spinal-bulbar muscular atrophy (SBMA), myotonic dystrophy (DM), fragile X syndrome, FRAXE mental retardation and spinocerebellar ataxia (SCA), Bruton's X-linked agammaglobulinemia (XLA), Bloom's syndrome (BS), craniofrontonasal syndrome (CFNS) and idiopathic pulmonary fibrosis (IPF).

[0080] amplification The method requires the amplification of a target nucleic acid sequence comprising a NOI from a first sample and a second sample. This requires that a set of primers is provided. The set of primers consists of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence comprising a NOI as known in the art.

[0081] When the set of primers is used in PCR, such as real-time PCR, in the presence of the target nucleic acid sequence, it can prime the amplification of the target nucleic acid using reagents that are otherwise known in the art. Such reagents are well known to those skilled in the art and are described, for example, in Sambrook J et al. 2000.Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Habor Laboratory Press. The reaction then produces an amplicon. When the amplification is carried out in the presence of a first sample, the produced amplicon is named as a first amplicon. When the amplification is carried out in the presence of a second (or reference) sample, the produced amplicon is named as a second amplicon or a reference amplicon.

[0082] To provide a set of primers specific for a target nucleic acid sequence, the primers comprise a sequence that is identical to a stretch of the target nucleic acid sequence. In particular, the 3' end of the first and second primers may comprise a sequence of at least 15 nucleotides that is identical to a stretch of the target nucleic acid sequence, including the reference or variant sequence, except for at most one mismatch.

[0083] The exact length of the primer sequence identical to the stretch of the target nucleic acid sequence can be adjusted to arrive at a primer with a melting temperature useful for PCR amplification. The melting temperature of the primer depends on several factors, but in particular on the GC content and length. Since the primer must preferably be identical to the stretch of the target nucleic acid sequence containing the mutant sequence (or the sequence complementary to the mutant sequence), there are restrictions on the specific sequence of the primer. Therefore, the melting temperature can be adjusted by adjusting in particular the length of the primer. The skilled person is fully capable of designing primers with appropriate melting temperatures, and useful software for this purpose is publicly available.

[0084] Thus, the 3' end of the primer may comprise a sequence of at least 15 nucleotides, such as at least 20 nucleotides, at least 25 nucleotides, such as in the range of 15-50 nucleotides, such as in the range of 20-40 nucleotides, that is identical to the target nucleic acid sequence including the variant sequence except for up to one mismatch.

[0085] In one embodiment of the invention, the first and / or second primer consists of a sequence of at least 15 nucleotides, such as at least 20 nucleotides, such as at least 25 nucleotides, such as in the range of 15-50 nucleotides, such as in the range of 20-40 nucleotides, that is identical to the target nucleic acid sequence including the variant sequence except for up to one mismatch.

[0086] As mentioned above, the sequence of the primer comprises or consists of the same (or complementary) sequence as the target nucleic acid sequence, including the mutant sequence, except for at most one mismatch.In some embodiments, there is one mismatch in one or both of the first and second primers, because it can further improve the specificity of the assay.In particular, the mismatch may be located at the 2nd, 3rd or 4th position from the 3' end of the primer.

[0087] In some embodiments, it may be useful to design primers that hybridize to the target nucleic acid sequence outside the region corresponding to the variant sequence. For example, if the target nucleic acid sequence is a microsatellite and the variant sequence differs from the reference sequence by the number of nucleotide repeats, the primers preferably hybridize upstream and downstream of the region consisting of tandem repeats, since amplification would otherwise be non-specific and would result in various amplicons of different lengths.

[0088] The primer may comprise at least one hydrophobic nucleotide as described herein.

[0089] The amplification step can be PCR, such as real-time PCR. In some embodiments, the amplification (e.g., PCR or real-time PCR) reaction is an asymmetric reaction. Asymmetric means that the reaction is directed to amplifying one strand of the template more than the other. This is accomplished by providing different amounts of the primers of the primer pair.

[0090] The first primer hybridizes to the first strand of the target nucleic acid sequence, which contains the NOI sequence. The second primer hybridizes to the second strand of the target nucleic acid sequence, which is complementary to the NOI sequence. In the latter step, the presence of the mutant sequence is detected by analyzing the melting profile of the amplicon, which contains a sequence identical to the NOI sequence, in the presence of a reporter probe, as described below. As shown in the examples, the analysis of the melting profile is facilitated if the strand of the amplicon to which the reporter oligonucleotide can hybridize is present in greater amounts than the strand of the amplicon that is complementary to the reporter oligonucleotide. Thus, in a preferred embodiment, the amplification step is asymmetric, i.e. it is carried out with a greater amount of the second primer than the first primer. This directs the amplification to generate more of the strand of the amplicon to which the reporter oligonucleotide hybridizes.

[0091] Melting analysis (including HRM analysis) Melting analysis, especially HRM (high resolution melting) analysis, is based on the analysis of the melting characteristics of the formed heteroduplex amplicons, especially the transition profile from double-stranded phase to single-stranded phase. The melting profile of amplicons depends on their guanine-cytosine content, length, sequence, and heterozygosity. Changes in nucleotide sequence result in the formation of heteroduplexes that change the shape of the melting curve compared to the melting profile of wild type.

[0092] When the first amplicon and the second amplicon are obtained, for example, through the use of asymmetric amplification reaction, the method includes carrying out melting analysis, in particular HRM analysis, to obtain a melting profile (for example, HRM profile) for each amplicon. A first melting profile (or a first HRM profile) is obtained for the first amplicon, and a second melting profile (or a second HRM profile), also called reference profile (or reference HRM profile), is obtained for the second amplicon. The melting profiles (or HRM profiles) are then compared as described in detail below.

[0093] Each melting (e.g., HRM) profile is characterized by a melting curve: a first melting (e.g., HRM) profile of a first amplicon is characterized by a first melting curve, and a second (or reference) melting (e.g., HRM) profile of a second amplicon is characterized by a second (or reference) melting curve.

[0094] The melting (e.g., HRM) analysis of each amplicon includes hybridization of a reporter oligonucleotide, which comprises at least one fluorophore and one quencher, to one strand, e.g., the second strand, of each amplicon (having a sequence identical to the NOI sequence). The reporter oligonucleotide is described in more detail below. After hybridization, the signal emitted by the fluorophore is detected to obtain a first and a second melting curve, as otherwise known in the art. The method may further include a step of transforming the melting curve before further analysis. For example, the melting curve can be transformed into a negative first derivative curve, which is then compared.

[0095] The first melting (e.g., HRM) profile and the second melting (e.g., HRM) profile are then compared. If the first sample contains a mutant sequence, then the first melting curve (and optionally the difference curve, and the derivative from the melting curve) is different from the second melting curve (and optionally the derivative from it) corresponding to the reference sample. Detection of the difference between the first and second melting (e.g., HRM) profiles thus indicates that the first sample contains a mutant sequence.

[0096] The difference between melting (e.g., HRM) profiles can be the difference in the shape of the melting curve or difference curve, or the shape of the derivative of the melting curve. For example, in some embodiments, the first melting curve has a different shape from the reference melting curve. Typically, the derivative of the melting curve is a normal curve (or a bell-shaped curve) with a maximum value. The slope of the first melting curve can be less "sharp" than the slope of the second curve, which results in a change in the shape of the first melting curve compared to the second melting curve. The slope of the derivative of the first melting curve can be "sharper" than the slope of the derivative of the second curve, e.g., the absolute value of the slope is greater for the derivative of the first melting curve than the absolute value of the slope of the derivative for the second melting curve, at least at its inflection point(s). To determine differences between melting (e.g., HRM) profiles, the analysis may include a step of obtaining difference curves, in which one of the melting curves is set as a reference and subtracted from the other melting curve, preferably the melting curve of the reference sample is set as a reference and subtracted from the melting curve of the mutant sample.

[0097] The two curves (or difference curves or derivatives) are given by T The distance D T is determined for each temperature and the maximum distance maxD T The absolute value of indicates a difference between the two curves if it is greater than a predefined threshold.

[0098] The skilled artisan knows how to determine an appropriate value for the threshold value, which makes it possible to distinguish between subjects exhibiting microsatellite instability and normal subjects. T value. D TThe value is the difference, e.g., negative difference or positive difference, between the melting profile obtained for the reference (or wild type) and the melting profile obtained for the mutant sequence(s). To determine an appropriate threshold, melting profiles of many subjects (typically 100-500 patients) known to have or not have microsatellite instability are established. Using these melting profiles, a threshold is established as a value that allows the desired or appropriate discrimination between wild type and mutant sequences.

[0099] Thus, the difference between the analyzed melting profiles is calculated and compared to a threshold value; this difference can therefore be a numerical difference, which can be compared to a numerical threshold value. If this difference between the analyzed melting profiles, e.g. HRM profiles, is greater than a threshold value, the sample from which the first melting profile is determined is classified as containing a mutant sequence. If the target nucleic acid sequence is a microsatellite, the subject from which the sample is obtained is therefore considered to have microsatellite instability. If the difference between the analyzed melting profiles, e.g. HRM profiles, is lower than a threshold value, the sample is classified as containing a wild-type sequence. If the target nucleic acid sequence is a microsatellite, the subject from which the sample is obtained is therefore considered to not have microsatellite instability.

[0100] One option is to determine the area between the curves. If the area is greater than a predefined threshold, the first and second melting curves are considered to be different. Again, those skilled in the art know how to set the value for the threshold.

[0101] Other methods of characterizing the difference between two melting curves or their difference curves or their derivatives will be apparent to one of skill in the art.

[0102] Any of the above differences between the first and second melting curves or their difference curves or derivatives of the first and second melting curves indicate differences between the first and second HRM profiles and thus indicate the presence of a mutant sequence in the first sample.

[0103] In some embodiments, the first and second melting curves and / or their derivatives and / or difference curves are normalized. For example, at or near the lowest temperature at which melting is measured, such as within 0.5-20° C. of the lowest temperature, the curves are normalized to a first value, and at or near the highest temperature at which melting is measured, such as within 0.5-20° C. of the highest temperature, the curves are normalized to a second value. For example, the first value is 1 and the second value is 0.

[0104] To better visualize the differences between the curves, the melting analysis, e.g., HRM analysis, may include a step of standard normalization, which corresponds to aligning the curves on the y-axis based on the average dye intensity in the initial and final normalized regions.

[0105] Bilinear normalization can also be applied to each curve (melt curve, difference graph or derivative). A first linear function is fitted to the initial dye intensities and used as the upper end of the final correction scale. A second linear function is fitted to the final dye intensities and used as the lower end of the correction scale.

[0106] The melting analysis, e.g., HRM analysis, may also or alternatively include a step of applying a temperature adjustment (temperature shift) in relative fluorescence units (RFU) to the curve (melting curve or difference curve). Such a step can also reduce the risk of false positives. In a preferred embodiment, the melting analysis includes a step of applying at least a temperature adjustment in relative fluorescence units to the melting curve.

[0107] Thus, in some embodiments, comparing the first profile to a reference profile comprises the steps of: i) aligning the first and second melting curves at a given fluorescence intensity along a temperature axis, thereby nullifying the difference in melting temperature between the first and second melting curves at said fluorescence intensity; ii) determining the difference in the signal emitted by the fluorophore between the first and second melting curves; and iii) comparing the difference determined in ii) to a threshold, wherein a difference greater than the threshold indicates that the first sample contains the variant sequence and a difference less than the threshold indicates that the first sample contains the reference sequence. It comprises or consists of:

[0108] The threshold is determined as described herein above. The difference is a numerical difference compared to the numerical threshold as described above. Thus, in step iii), if the threshold is a positive threshold, a difference greater than the threshold indicates that the first sample contains a mutant sequence. If the threshold is a negative threshold, a difference less than the threshold indicates that the first sample contains a mutant sequence.

[0109] In some embodiments, the method disclosed herein does not include a step of determining the exact length and / or sequence of the variant sequence. This is because it is often not necessary to determine the exact length and / or sequence of the variant sequence to determine that the individual in which the variant sequence is found suffers from a disease or disorder as described herein. Rather, it is often sufficient to establish that there is a difference - the exact nature of the difference is not always important. In a preferred embodiment, the method disclosed herein does not include a step of determining the exact length and / or sequence of the variant sequence. This is because the detection of the variant sequence may be sufficient to select a sample for further analysis; typically, it is not necessary to determine the exact length and / or sequence of the variant sequence.

[0110] Reporter Oligonucleotides The method requires a reporter oligonucleotide to carry out HRM analysis. The reporter oligonucleotide comprises at least a first fluorophore and at least a first quencher. These are useful for melting analysis such as HRM analysis. Preferably, the reporter oligonucleotide comprises a first fluorophore at least at its 5' end or at least at its 3' end or within 4 nucleotides from the 5' end or 3' end. The reporter oligonucleotide preferably comprises a first quencher at least at its 5' end or at least at its 3' end or within 4 nucleotides from the 5' end or 3' end. Thus, the fluorophore and quencher may be located at the 5' end or at the 3' end or within 4 nucleotides from the 5' end or 3' end, but are not necessarily located at the last nucleotide of the reporter oligonucleotide. Preferably, if the first fluorophore is located at the 5' end or within 4 nucleotides from the 5' end, the first quencher is not located at the 5' end or within 4 nucleotides from the 5' end. Instead, it is located at the 3'-end or within 4 nucleotides from the 3'-end, or within the internal region of the reporter. Conversely, if the first fluorophore is located at the 3'-end or within 4 nucleotides from the 3'-end, the first quencher is not located at the 3'-end or within 4 nucleotides from the 3'-end. Instead, it is located at the 5'-end or within 4 nucleotides from the 5'-end, or within the internal region of the reporter. Here, the term internal region refers to the region of the reporter oligonucleotide that does not include the 5-terminal nucleotide at each end. Preferably, the first fluorophore and the first quencher are not adjacent to each other.

[0111] Useful fluorophores and quenchers are readily available to one of skill in the art, and one would have no difficulty in selecting them to practice the present methods.

[0112] The reporter oligonucleotide is used in melting analysis (or HRM analysis). Therefore, the sequence of the reporter oligonucleotide is subject to some constraints that depend on the sequence it hybridizes to and detects. The reporter oligonucleotide comprises a hybridization sequence H, which is for example identical to the NOI and hybridizes to a strand complementary to the NOI. In other words, the reporter oligonucleotide hybridizes to the strands of the first and second amplicons that contain the NOI. In an embodiment in which the NOI comprises a repeat, the reporter oligonucleotide therefore also comprises the repeat, i.e. the hybridization sequence H also comprises the repeat, as further detailed below.

[0113] The reporter oligonucleotide may further comprise additional nucleotides in the hybridization sequence in addition to the sequence identical to the NOI. For example, as shown in the following examples, this may be particularly important when the target nucleic acid is a microsatellite or when the variant sequence is expected to contain an insertion. When the microsatellite has M tandem repeats with a total length of n nucleotides in the reference sequence, the hybridization sequence of the reporter oligonucleotide preferably has M" tandem repeats, where M"≧M+1, preferably M"≧M+1 or M"≧M+2. When the tandem repeats are mononucleotide repeats, the hybridization sequence has a length of n" nucleotides, where n"≧n+1, preferably n"≧n+1 or n"≧n+2. In other words, the hybridization sequence may comprise at least one or two additional nucleotides. This allows for more sensitive discrimination of longer variant and reference sequences.

[0114] In some embodiments, particularly where the NOI is a microsatellite, the hybridization sequence of the reporter oligonucleotide preferably comprises a sequence consisting of repeats that are identical or complementary to the repeats of the NOI, and may advantageously also comprise a terminal sequence, herein referred to as a helper sequence, which hybridizes to the first and second amplicons immediately upstream or downstream of the repeats when the hybridization sequence hybridizes to the repeats. In other words, the hybridization sequence of the reporter oligonucleotide preferably comprises a repeat and further comprises 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, which hybridize to the NOI or its complementary strand immediately upstream or downstream of the repeat sequence. The helper sequence thus enables the hybridization sequence to hybridize to said repeat sequence when hybridizing to the first and second amplicons. The helper sequence often has a higher affinity for its complementary sequence than the hybridization sequence for its complementary sequence. In other words, the helper sequence often hybridizes to its complementary sequence faster than the hybridization sequence hybridizes to its complementary sequence.

[0115] The helper sequence can be at the 3' or 5' end. Preferably, the reporter oligonucleotide contains a 5' end helper sequence that hybridizes to the first and second amplicons immediately upstream or downstream of the repeat when the hybridization sequence hybridizes to the repeat. The helper sequence facilitates the distinction between melting profiles, as shown in Example 13. A helper sequence is called strong if it results in an increase in the Tm of the hybridization sequence when the helper sequence hybridizes to the high first or second amplicon. Those skilled in the art are well aware that the hybridization between two sequences is strong when the sequence contains a higher percentage of G / C compared to the two hybridized sequences that contain a higher percentage of A / T; thus, a helper sequence that contains a higher percentage of G / C than A / T is stronger than a helper sequence that contains a lower percentage of G / C than A / T. It is also known that longer sequences give stronger hybridization than shorter sequences, and therefore the strength of the helper sequence can be adjusted by increasing the length of hybridization to the first and / or second amplicon. Increasing the strength of the helper sequence can be a good way to facilitate the discrimination of variant and reference sequences. Preferably, the helper sequence does not contain repeats, even in the embodiment in which the NOI contains repeats.

[0116] In particular, in some embodiments where the target nucleic acid sequence is a microsatellite, the helper sequence increases the Tm of the hybridization sequence by at least 5° C., such as at least 6° C., for example at least 7° C., such as at least 8° C., for example at least 9° C., for example at least 10° C., for example at least 11° C., such as at least 12° C., for example at least 13° C., for example at least 14° C., for example at least 15° C., or more, compared to the Tm of the same hybridization sequence without the helper sequence. The Tm of the hybridization sequence comprising the helper sequence may thus be 5 to 25° C. higher, such as 10 to 20° C. higher, for example 12.5 to 17.5° C. higher, than the Tm of the hybridization sequence without the helper sequence.

[0117] Reporter oligonucleotide may comprise a second quencher.Preferably, the second quencher is located in non-terminal region, i.e., in the internal region of reporter oligonucleotide.In other words, the second quencher is not located at the 5'-end or the 3'-end, or within 4 nucleotides from the 5'-end or the 3'-end.In some embodiments, the inclusion of the second quencher facilitates the distinction between variant sequence and reference sequence.

[0118] Any reporter oligonucleotide that can enable melting analysis, particularly HRM analysis, i.e., any reporter oligonucleotide having the above-mentioned characteristics can be used, but some reporter oligonucleotides are particularly advantageous.The reporter oligonucleotide contains hydrophobic nucleotides at certain positions and is particularly interesting.As can be seen from the examples, the reporter oligonucleotides that contain such hydrophobic nucleotides increase the sensitivity of the method.

[0119] Thus, in some embodiments, in addition to the features described above, the reporter oligonucleotide comprises at least one hydrophobic nucleotide located at or within 10 nucleotides from its 5' end, and / or the reporter oligonucleotide comprises at least one hydrophobic nucleotide located at or within 10 nucleotides from the 3' end.

[0120] Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator. has.

[0121] The backbone monomer unit X can be any backbone monomer unit described herein in the section "Backbone Monomer Units" below.

[0122] The intercalator Q can be any intercalator described herein below in the section "Intercalators."

[0123] Hydrophobic nucleotides that are useful in the context of the present disclosure are described in detail in International Patent Application WO2017 / 045689, in particular in the section entitled "Hydrophobic Nucleotides" on page 30, lines 2-25.

[0124] In some embodiments, the reporter oligonucleotide has the general structure: 5'-(N) a -Z-(N) d- Z-(N) e -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and the total number of nucleotides or nucleotide analogs is at least 10; and a and b are each an integer ranging from 0 to 4; and d and e are each an integer ranging from 1 to 19; and a+b+d+e is at least 10) having; (N) a -(N) d -(N) e -(N) b is identical to the reference sequence.

[0125] In other embodiments, the reporter oligonucleotide has the general structure 5'-(N) a -Z-(N) f- Z-(N) g-Z-(N) h -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and f, g and h are individually integers ranging from 1 to 18; and a+b+f+g+h is at least 10 and at most 50; and (N) a -(N) f -(N) g -(N) h -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) i -Z-(N) j -Z-(N) k -Z-(N) l -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and i, j, k and l are integers ranging from 1 to 17; and a+b+i+j+k+l are individually at least 10 and at most 50; and (N) a -(N) i -(N) j -(N) k -(N) l -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) m -Z-(N) n -Z-(N) o -Z-(N) p -Z-(N) q -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and m, n, o, p and q are individually integers ranging from 1 to 16; and a+b+m+n+o+p+q is at least 10 and at most 50; and (N) a -(N) m -(N) n -(N) o -(N) p -Z-(N) q -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) has.

[0126] In some embodiments, the reporter oligonucleotide is for detection of a microsatellite. In such embodiments, the hybridization sequence comprises at least a tandem repeat. Preferably, the hybridization sequence also comprises a helper sequence consisting of 1-20 nucleotides immediately upstream or downstream of the tandem repeat, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides immediately upstream or downstream of the tandem repeat, preferably with at least one hydrophobic nucleotide inserted as described herein. The helper sequence may consist of at least one of 1, such as 2, 3, such as 4, 5, such as 6, such as 7, such as 8, 9, such as 10, such as 10-20, such as 20-50, more than 50, etc. nucleotides. For example, the helper sequence consists of 15 or more nucleotides. Thus, in some embodiments, the helper sequence consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20 or more nucleotides, such as 25, 30, 35, 40, 45 or 50 or more nucleotides. For example, the helper sequence consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. For example, the helper sequence consists of 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides, such as 9 nucleotides.

[0127] In some embodiments, reporter oligonucleotides are used for detection of microsatellites, such as BAT25, BAT26, NR21, NR22, NR24, or MONO27, as described above.

[0128] Intercalators The term intercalator in the context of the present method refers to any molecular moiety that comprises at least one essentially planar conjugated system that can be stacked simultaneously with the nucleobases of a nucleic acid.Preferably, the intercalator consists essentially of at least one essentially planar conjugated system that can be stacked simultaneously with the nucleobases of a nucleic acid.

[0129] Intercalators contain at least one π (phi) electron system, which according to the present invention can interact with other molecules that contain π electron systems. These interactions can contribute to the hydrophobic interactions of the intercalators in a positive or negative manner. Hunter and Sanders (1990) J. Am Chem. Soc. 112:5525-5534 propose a range of different orientations and conditions in which two π electron systems can interact positively with each other.

[0130] Preferably, the intercalator comprises a chemical group selected from the group consisting of polyaromate and heteropolyaromate, even more preferably, the intercalator consists essentially of polyaromate or heteropolyaromate. Most preferably, the intercalator is selected from the group consisting of polyaromate and heteropolyaromate.

[0131] The polyaromate or heteropolyaromate according to the present invention may consist of any suitable number of rings, such as 1, for example 2, 3, for example 4, 5, for example 6, 7, for example 8, more than 8, etc. Furthermore, the polyaromate or heteropolyaromate may be substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocyclic, aryl, heteroaryl, carboxyl, carboalkoxyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, carbonyl and amido.

[0132] Thus, the intercalator Q may be, for example, an intercalator selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, picene, chrysene, naphthacene, acridone, benzanthracene, stilbene, oxalo-pyridocarbazole, azidobenzene, porphyrin, psoralen, and any of the aforementioned intercalators substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocyclic, aryl, heteroaryl, carboxyl, carboalkoxyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl and / or amide.

[0133] Preferably, the intercalator is selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, picene, chrysene, naphthacene, acridone, benzanthracene, stilbene, oxalo-pyridocarbazole, azidobenzene, porphyrin and psoralen.

[0134] In a preferred embodiment, the intercalator is selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, phenanthrene, chrysene, naphthacene, benzanthracene, stilbene, and porphyrin.

[0135] In another preferred embodiment, the intercalator comprises pyrene or pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4(1H)-one or 7,9-dimethyl-pyrido[3',2',4,5]thieno[3,2-d]pyrimidin-4(3H)-one. The intercalator also comprises pyrene or pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4(1H)-one or 7,9-dimethyl-pyrido[3',2',4,5]thieno[3,2-d]pyrimidin-4(3H)-one.

[0136] The hydrophobic nucleotide may include other intercalators, in particular those described in WO2017 / 045689, page 30, line 26 to page 40, line 4, section entitled "Intercalators", or in International Patent Application WO03 / 052132, page 46, line 10 to page 54, line 13, section entitled "Intercalators".

[0137] Backbone Monomer Unit X may also be a backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog. Backbone monomer unit of a nucleotide or nucleotide analog herein refers to the part of the nucleotide that is involved in incorporation into the backbone of a nucleic acid or nucleic acid analog. Backbone monomer unit (X) is preferably covalently linked to a linker (Y) that is covalently linked to an intercalator. Any suitable backbone monomer unit can be used to incorporate an intercalator into an oligonucleotide analog. Any type of linker that connects the backbone monomer unit and the intercalator can also be used. In addition, the backbone monomer unit may include one or more leaving groups, protecting groups and / or reactive groups, which can be removed or modified in any manner during or following the synthesis of an oligonucleotide or oligonucleotide analog that includes the backbone monomer unit.

[0138] The backbone monomer unit may be any suitable backbone monomer unit. In one embodiment, the backbone monomer unit may be, for example, DNA, RNA, PNA, HNA, XNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, aL-ribo-LNA, aL-xylo-LNA, bD-xylo-LNA, aD-ribo-LNA, [3.2.1]-LNA, bicyclo-DNA, 6-amino-bicyclo-DNA, 5-epi-bicyclo-DNA, α-bicyclo-DNA, tricyclo-DNA, bicyclo[4.3.0]-DNA, bicyclo[3.2 The backbone monomer units of the nucleotides may be selected from the group consisting of nucleotides such as, but not limited to, phosphorothioates, methyl phosphonates, phosphoramidites, phosphorodithioates, phosphoroselenoates, phosphotriesters, and phosphoboranoates, as well as backbone monomer units of the nucleotides such as, but not limited to, phosphorothioates, methyl phosphonates, phosphoramidites, phosphorodithioates, phosphoroselenoates, phosphotriesters, and phosphoboranoates. In addition, non-phosphorus-containing compounds may be used for linking to nucleotides and amide-containing linking groups such as, but not limited to, methyliminomethyl, formacetate, thioformacetate, and the like.

[0139] A range of backbone monomer units are described in International Patent Application WO2017 / 045689, in the section entitled "Backbone Monomer Units" on page 40, line 5 to page 56, line 3, and in International Patent Application WO03 / 052132, in the section entitled "Backbone Monomer Units" on page 24, line 27 to page 43, line 14. These also describe a variety of different backbone monomer units of nucleotides and nucleotide analogues useful in the present invention and how they are connected to the nucleobase via linkers attached at one or two positions of the backbone monomer unit.

[0140] Linker The linker of the intercalator nucleotide is the moiety that connects the intercalator and the backbone monomer of the hydrophobic nucleotide, preferably covalently bonds the intercalator and backbone monomer units. The linker may include one or more atom(s) or bond(s) between the atoms.

[0141] According to the definitions of the scaffold and the intercalator given herein above, the linker is the shortest path connecting the scaffold and the intercalator. If the intercalator is directly connected to the scaffold, the linker is a bond.

[0142] Linkers usually consist of a chain of atoms or a branched chain of atoms. The chain may be saturated or unsaturated. The linker may also be a ring structure with or without covalent bonds.

[0143] Useful linkers are described in detail in International Patent Application WO2017 / 045689, in particular the section entitled "Linkers" from page 56, line 5 to page 59, line 10, and in WO03 / 052132, the section entitled "Linkers" from page 54, line 15 to page 58, line 7.

[0144] Kit of parts Also provided herein is a kit-of-parts for detecting the presence of a variant sequence in a target nucleic acid sequence comprising a nucleotide(s) of interest (NOI) consisting of two strands and preferably comprising repeats, said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said kit-of-parts comprising: a) a reporter oligonucleotide that preferably comprises a first fluorophore at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end. (The reporter nucleotide is a sequence of 10 to 50 nucleotides, preferably 15 to 50 nucleotides, in which 2 to 10 hydrophobic nucleotides are inserted; and The reporter oligonucleotide comprises a hybridization sequence H, and the hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid; and the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence being free of repeats and capable of hybridizing to the first and second amplicons when the hybridization sequence hybridizes thereto; and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence; Includes.

[0145] In this specification: a) a reporter oligonucleotide, which may be any of the reporter oligonucleotides described herein below in the section "Reporter Oligonucleotides." b) a set of primers consisting of a first primer and a second primer, which may be any set of primers described herein in the section "Amplification" above; A kit of parts is also provided, including:

[0146] Kits of parts are particularly useful for carrying out the present methods.

[0147] Thus, also provided herein is a kit-of-parts for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), said target nucleic acid sequence consisting of a variant or reference sequence, said kit-of-parts comprising: a) a reporter oligonucleotide that preferably comprises a first fluorophore at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end. (The reporter oligonucleotide is a sequence of 10-50 nucleotides having 2-10 hydrophobic nucleotides inserted therein, the reporter oligonucleotide including a hybridization sequence H), (at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid containing the reference sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid; and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid; Includes.

[0148] In addition to the reporter oligonucleotide and the primer set, the kit of parts may also include additional components.For example, the kit of parts may further include PCR reagents.The kit of parts may also include detection probes, such as probes that allow real-time detection of the generation of PCR products.

[0149] Said kit is particularly useful for carrying out the methods described herein, in particular for detecting variant sequences of target nucleic acid sequences, preferably including repeats, such as microsatellites, in which the wild type or reference sequence is 15 nucleotides or more, as detailed herein above.

[0150] Reporter Oligonucleotides As described hereinabove in detail, this method requires reporter oligonucleotide to carry out melting analysis, for example to carry out HRM analysis.Without being bound by theory, the inventors have found that reporter oligonucleotides that comprise hydrophobic nucleotides are particularly useful for carrying out this method, because they may increase the sensitivity of the assay.Such reporter oligonucleotides are therefore also disclosed herein.

[0151] Also provided herein is a reporter oligonucleotide that is composed of two strands and is capable of hybridizing to one strand of a target nucleic acid that comprises a nucleotide(s) of interest (NOI), preferably comprising a repeat, said reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end, said reporter oligonucleotide being a sequence of 10-50 nucleotides, preferably in the range of 15-50 nucleotides, interrupted by 2-10 hydrophobic nucleotides, said reporter oligonucleotide comprising a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid; The hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or its 3' end, said helper sequence not comprising the repeat and capable of hybridizing to the second strands of the first and second amplicons when the hybridization sequence hybridizes to the amplicon.

[0152] Also provided herein is a reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end, wherein the reporter oligonucleotide is a sequence in the range of 10-50 nucleotides interspersed therewithin, with between 2 and 10 hydrophobic nucleotides, wherein the reporter oligonucleotide comprises a hybridization sequence H; at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and The hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid.

[0153] Reporter oligonucleotide may comprise a second quencher.Preferably, the second quencher is located in a non-terminal region, i.e., in the internal region of reporter oligonucleotide.In other words, the second quencher is not located at the 5'-end or the 3'-end, or within 4 nucleotides from the 5'-end or the 3'-end.

[0154] The backbone monomer unit X can be any backbone monomer unit described herein in the section "Backbone Monomer Units" above.

[0155] The intercalator Q can be any intercalator described herein in the section "Intercalators" above.

[0156] Hydrophobic nucleotides that are useful in the context of the present disclosure are described in detail in International Patent Application WO2017 / 045689, in particular in the section entitled "Hydrophobic Nucleotides" on page 30, lines 2-25.

[0157] In some embodiments, the reporter oligonucleotide has the general structure: 5'-(N) a -Z-(N) d- Z-(N) e -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and the total number of nucleotides or nucleotide analogs is at least 10; and a and b are each an integer ranging from 0 to 4; and d and e are each an integer ranging from 1 to 19; and a+b+d+e is at least 10) having; (N) a -(N) d -(N) e-(N) b is identical to the reference sequence.

[0158] In another embodiment, the reporter oligonucleotide has the general structure 5'-(N) a -Z-(N) f- Z-(N) g -Z-(N) h -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and f, g and h are individually integers ranging from 1 to 18; and a+b+f+g+h is at least 10 and at most 50; and (N) a -(N) f -(N) g -(N) h -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) i -Z-(N) j -Z-(N) k -Z-(N) l -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and i, j, k and l are integers ranging from 1 to 17; and a+b+i+j+k+l are individually at least 10 and at most 50; and (N) a -(N) i -(N) j -(N) k -(N) l -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) m -Z-(N) n -Z-(N) o -Z-(N) p -Z-(N) q -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in claim 1; and a and b are each an integer ranging from 0 to 4; and m, n, o, p and q are individually integers ranging from 1 to 16; and a+b+m+n+o+p+q is at least 10 and at most 50; and (N) a -(N) m -(N) n -(N) o -(N) p -Z-(N) q -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) has.

[0159] The reporter oligonucleotide may comprise an overhang, i.e., it may comprise a nucleotide at one of its ends that does not hybridize to the target nucleic acid sequence. The overhang may be at the 3' end or the 5' end. Preferably, the reporter oligonucleotide comprises a 5' end sequence that forms a 5' overhang with respect to the strands of the first and second amplicons to which the hybridization sequence can hybridize.

[0160] The reporter oligonucleotide comprises at least a first fluorophore and at least a first quencher. These are useful for melting analysis and / or HRM analysis. Preferably, the reporter oligonucleotide comprises a first fluorophore at least at its 5'-end or at least at its 3'-end or within 4 nucleotides from the 5'-end or 3'-end. The reporter oligonucleotide preferably comprises a first quencher at least at its 5'-end or at least at its 3'-end or within 4 nucleotides from the 5'-end or 3'-end. Thus, the fluorophore and quencher may be located at the 5'-end or 3'-end or at the 5'-end or within 4 nucleotides from the 3'-end, but not necessarily at the last nucleotide of the reporter oligonucleotide. Preferably, if the first fluorophore is located at the 5'-end or within 4 nucleotides from the 5'-end, the first quencher is not located at the 5'-end or within 4 nucleotides from the 5'-end. Instead, it is located at the 3'-end or within 4 nucleotides from the 3'-end or within an internal region of the reporter. Conversely, if the first fluorophore is located at the 3'-end or within 4 nucleotides from the 3'-end, the first quencher is not located at the 3'-end or within 4 nucleotides from the 3'-end.Instead, it is located at the 5'-end or within 4 nucleotides from the 5'-end, or within the internal region of the reporter.The term internal region as used herein refers to the region of the reporter oligonucleotide that does not include the 5'-end nucleotide at each end.Preferably, the first fluorophore and the first quencher are not adjacent to each other.

[0161] Useful fluorophores and quenchers are readily available to one of skill in the art, and one would have no difficulty in selecting them to practice the present methods.

[0162] Reporter oligonucleotides are used to perform melting analysis, such as HRM analysis. Therefore, the sequence of the reporter oligonucleotide is subject to some constraints, depending on the sequence it hybridizes to and is detected. The reporter oligonucleotide comprises a hybridization sequence H, which is, for example, identical to the NOI and hybridizes to the strand complementary to the NOI. In embodiments in which the NOI comprises repeats, the reporter oligonucleotide therefore also comprises the repeats, i.e. the hybridization sequence H also comprises the repeats, as further detailed below.

[0163] The reporter oligonucleotide further comprises additional nucleotides in the hybridization sequence in addition to the sequence identical to the NOI. For example, as shown in the examples below, this may be particularly important when the target nucleic acid is a microsatellite. When the microsatellite has M tandem repeats in the reference sequence with a total length of n nucleotides, the hybridization sequence of the reporter oligonucleotide preferably has M" tandem repeats, where M"≧M+1, preferably M"≧M+1 or M"≧M+2. Thus, when the tandem repeats are mononucleotide repeats, the hybridization sequence has a length of n" nucleotides, where n"≧n+1, preferably n"≧n+1 or n"≧n+2. In other words, the hybridization sequence may comprise at least one or two additional nucleotides. This allows for more sensitive discrimination of the variant and reference sequences, especially when the variant sequence contains an insertion.

[0164] In some embodiments, particularly where the NOI is a microsatellite, the hybridization sequence of the reporter oligonucleotide preferably comprises a sequence consisting of repeats which are identical to or complementary to the repeats of the NOI and may advantageously also comprise terminal sequences, such as one or two terminal sequences, referred to herein as helper sequence(s), which hybridize to the strands of the first and second amplicon immediately upstream or downstream of the repeats when the hybridization sequence hybridizes to the repeats. In other words, the hybridization sequence of the reporter oligonucleotide preferably comprises a repeat and further comprises 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides which hybridize to the NOI or its complementary strands immediately upstream or downstream of the repeated sequence.

[0165] The helper sequence can be at the 3' or 5' end. Preferably, the reporter oligonucleotide contains a 5' end helper sequence that hybridizes to the strands of the first and second amplicons immediately upstream or downstream of the repeat when the hybridization sequence is hybridized to the repeat. The helper sequence facilitates the distinction between melting profiles, as shown in Example 13. A helper sequence is called strong if it has a high Tm when hybridized to the first or second amplicon. Those skilled in the art are well aware that the hybridization between two sequences is strong when the sequence contains a higher percentage of G / C compared to the two hybridized sequences that contain a higher percentage of A / T; thus, a helper sequence that contains a higher percentage of G / C than A / T is stronger than a helper sequence that contains a lower percentage of G / C than A / T. It is also known that longer sequences confer stronger hybridization than shorter sequences, and therefore the strength of the helper sequence can be adjusted by increasing the hybridizing length to the first and / or second amplicon.

[0166] In some embodiments, the reporter oligonucleotide is useful for detecting mutant sequences indicative of microsatellite instability, i.e., the reference sequence comprises a microsatellite sequence. If the reference sequence comprises M tandem repeats of a microsatellite sequence having a total length of n nucleotides, the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1, and preferably n"≧n+1 or n"≧n+2.

[0167] The reporter oligonucleotide may comprise a second quencher.Preferably, the second quencher is located in a non-terminal region, i.e., in an internal region of the reporter oligonucleotide.In other words, the second quencher is not located at the 5'-end or the 3'-end, or within 4 nucleotides from the 5'-end or the 3'-end.

[0168] Useful fluorophores and quenchers are known in the art and readily available to the skilled artisan, who would have no difficulty selecting appropriate fluorophores and quenchers.

[0169] Also provided herein is the use of such reporter oligonucleotides comprising hydrophobic nucleotides in methods for detecting mutant nucleic acids, particularly their use in the methods described herein. Reporter oligonucleotides can also be used in methods for predicting the efficacy of treatment of a clinical condition, and in methods for predicting the presence of a clinical condition, particularly as described herein below.

[0170] Predicting the effectiveness of treatment for clinical conditions The present disclosure also relates to a method for predicting the efficacy of treating a clinical condition in an individual in need thereof with a given agent, wherein the efficacy of treating said clinical condition with said agent is related to the presence of a variant sequence.

[0171] Therefore, some mutations may suggest whether a particular drug is effective or not for treating an individual.In particular, certain mutations may suggest a particular response to a given drug treatment.For example, mutations may suggest whether an individual responds positively to said drug treatment, whether the individual's disease is resistant to a given drug, or whether the individual cannot tolerate a particular drug treatment.

[0172] A method for predicting the efficacy of a treatment for a clinical condition in an individual includes the steps of: a. Preparing a sample from said individual b. performing a method for detecting the presence of a variant sequence as described herein to determine whether the sample contains the variant sequence. may include The presence of the variant sequence indicates whether the agent will be effective in treating the clinical condition in the individual.

[0173] The clinical condition may be, for example, cancer, or any clinical condition, disease or disorder described herein. Many mutations have been identified that suggest whether a given cancer drug or drug combination will be effective in treating a particular cancer.

[0174] In some embodiments, the variant sequence is a microsatellite variant, as detailed herein above.

[0175] Predicting the presence of clinical conditions The methods are also useful for predicting the presence, or even diagnosing, of any clinical condition associated with a particular mutation in an individual.

[0176] Such a method comprises the steps of: a) preparing a sample from said individual; b) detecting the presence of a mutation associated with a clinical condition in the sample by carrying out a method for detecting a variant sequence as described herein. Well containing; The presence of the variant sequence is indicative of the individual suffering from the clinical condition.

[0177] Many clinical conditions are known to be associated with specific mutations, and a set of reporter oligonucleotides and primers can be designed to detect such mutations. The method is particularly useful for detecting conditions associated with microsatellite instability when used to detect mutant sequences of microsatellites, preferably with corresponding wild-type or reference sequences at least 15 nucleotides in length, as detailed above.

[0178] In one embodiment, the clinical condition is cancer, such as hereditary nonpolyposis colorectal cancer, hi another embodiment, the clinical condition is Lynch syndrome.

[0179] The method may further comprise treating said clinical condition. If a variant sequence indicative of the presence of a clinical condition is found, the individual is classified as suffering from said clinical condition, and the method may further comprise administering to said individual an effective amount of a therapeutic agent.

[0180] Working Example Example 1: Microsatellite instability does not necessarily result in a change in melting temperature. BAT25 assay using asymmetric PCR with primers hybridizing upstream and downstream of the region containing the mononucleotide repeat. The assay is for detecting mutant sequences in the BAT25 microsatellite. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The reporter oligonucleotide hybridizes to positions 142-171 of SEQ ID NO:1, has an overhang at its 3' end, and a helper sequence at its 5' end, which hybridizes upstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end, and a hydrophobic nucleotide within 10 nucleotides from the 3' end. Normal and tumor tissue DNA from CRC or endometrial samples from microsatellite instability patients at 1-2 ng / μL were tested. The initial normalization interval was 39-40°C, and the final normalization interval was 63-64°C. Two melting curves were obtained (FIG. 2A) and transformed into negative first derivative curves (FIG. 2B). The two curves were not identical when temperature was adjusted by applying bilinear normalization and applying a temperature shift intensity threshold of 0.1 RFU above the 0.05 RFU threshold (the maximum difference between the amplitudes of the two curves was approximately 0.07 RFU (absolute value), not shown). The difference of 0.07 is higher than the 0.05 RFU threshold set for this particular assay, and therefore the sample is classified as unstable for BAT25. The negative first derivative curve is shown in FIG. 2B. As can be seen, normal tissue has a T of 57.31° C. m and the tumor tissue had a T of 57.41°C. m Such small differences are usually inconsequential and could be due to different salt concentrations in the samples or instrument variability.

[0181] From this example, it can be concluded that the melting temperature does not necessarily change significantly from normal to mutated tumor samples, but the shape of the melting curve changes, which is more easily visualized when applying bilinear normalization and temperature shifts. The normal and tumor samples therefore have a T m Even very small differences in can be distinguished using the difference in shape between the HRM curves.

[0182] Example 2: Bilinear normalization NR22 assay using asymmetric PCR with primers hybridizing upstream and downstream of the region containing the mononucleotide repeat. The assay is for detecting mutant sequences in the NR22 microsatellite. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 143-172 of SEQ ID NO:4, has an overhang (2 nucleotides) at its 3' end, and a helper sequence (9 nucleotides; resulting in an increase in Tm of 14.2°C) at its 5' end, which hybridizes upstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end, and a hydrophobic nucleotide within 10 nucleotides from the 3' end. FFPE purified normal and tumor tissue DNA from microsatellite instability patients at 1-2 ng / μL was tested. Two melting curves were obtained (Figure 3). Figure 3A shows the melting curves without bilinear normalization or temperature shift applied, and Figure 3B shows the melting curves after bilinear normalization (no temperature shift).

[0183] FIG. 4 shows the difference in fluorescence D between the reference (fluorescence set to 0 at any given temperature; samples are from healthy cells of a patient) and tumor samples as a function of temperature. T This is also called a difference plot, difference curve or difference graph. Maximum difference maxD TIt is measured as the absolute value of the maximum amplitude of the difference curve. Data after applying standard normalization (Figure 4A) or after applying bilinear type normalization (without temperature shift) (Figure 4B) are shown.

[0184] This example shows that bilinear type normalization can compensate for different decreases in fluorescence confirmed before and after melting of different samples and helps to distinguish HRM profiles for normal (healthy) samples and tumor samples.

[0185] Example 3: Bilinear type normalization and temperature shift can reduce the differences between melting curves. NR24 assay using asymmetric PCR. The assay is for detecting mutant sequences in the NR24 microsatellite. The reporter oligonucleotide used in this example held a FAM fluorophore at the 5'-end and a BHQ-1 quencher at the 3'-end, as well as an internal quencher. The reporter oligonucleotide hybridized to positions 158 to 187 of SEQ ID NO: 5, had an overhang at its 3'-end, and a helper sequence (bringing about an increase in Tm at 14.8 °C) at its 5'-end, which hybridized upstream of the mononucleotide repeat. In addition, it contained a hydrophobic nucleotide within 10 nucleotides from the 5'-end and a hydrophobic nucleotide within 10 nucleotides from the 3'-end. Normal tissue DNA and tumor tissue DNA at 1 - 2 ng / μL from the same microsatellite stability patient were tested. Standard normalization was applied at an initial interval of 44 - 45 °C (set as value 1) and a final interval of 66 - 67 °C (set as value 0). The results are shown in Figure 5.

[0186] When bilinear type normalization and temperature shift intensity threshold were not applied, the difference between the curves was about 0.04 RFU (Figure 5A). When bilinear type normalization was applied but the temperature shift intensity threshold was not applied, the difference between the curves was about 0.02 RFU (Figure 5B). When bilinear type normalization and a temperature shift intensity threshold of 0.1 RFU were applied, the difference between the curves was about 0.01 RFU (Figure 5C).

[0187] From this example, it can be concluded that bilinear normalization and temperature shifting can reduce the difference between the melting curves of healthy and normal samples from microsatellite stability patients, which reduces the risk of false positives.

[0188] Example 4: A temperature shift intensity threshold can neutralize changes in melting temperature caused by different salt concentrations in DNA buffers. NR24 assay using asymmetric PCR. The assay is for detecting mutant sequences in NR24 microsatellites. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 158-187 of SEQ ID NO:5, has an overhang (2 nucleotides) at its 3' end, and a helper sequence at its 5' end (resulting in an increase in Tm of 14.8°C), which hybridizes upstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end, and a hydrophobic nucleotide within 10 nucleotides from the 3' end. Normal tissue DNA was diluted to 40ng / μL-2ng / μL with water and TE buffer, respectively. Initial normalization was performed in the region 44-45°C, and final normalization was performed in the region 66-67°C. The results are shown in Figure 6.

[0189] The maximum difference between the curves, maxD, when bilinear normalization was applied but no temperature shift was applied. T was approximately 0.07 RFU (Figure 6A). When bilinear normalization and a temperature shift with an intensity threshold of 0.1 RFU were applied, maxD T was approximately 0.01 RFU (Figure 6B).

[0190] From this example, it can be concluded that the application of a temperature shift can reduce the differences between two samples when the differences are caused by different salt concentrations in the DNA buffer. Thus, after bilinear normalization and application of a temperature shift, aliquots of one sample can produce identical HRM profiles even if the aliquots are obtained in different buffers. This significantly reduces the risk of false positive calls, since the assay is less vulnerable to variations in salt and buffer concentrations between the samples being compared.

[0191] Example 5: Temperature shift creates the possibility to use one universal reference sample. MONO27 assay using asymmetric PCR with primers hybridizing upstream and downstream of the region containing the mononucleotide repeat. The assay is for detecting mutant sequences in the MON27 microsatellite. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 300-333 of SEQ ID NO:6, has an overhang at its 3' end, and a helper sequence at its 5' end (resulting in an increase in Tm of 10.8°C), which hybridizes upstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end and a hydrophobic nucleotide within 10 nucleotides from the 3' end. FFPE purified normal tissue DNA from 16 different patients at 1-2 ng / μL was tested. The initial normalized interval was 44-45°C and the final normalized interval was 66-67°C.

[0192] FIG. 7A shows the HRM curves where bilinear normalization was applied but no temperature shift was applied. The maximum difference between the curves, maxD T was approximately 0.25 RFU. FIG. 7B shows the HRM curves to which bilinear normalization and temperature shift with an intensity threshold of 0.1 RFU were applied. T The RFU decreased to approximately 0.06.

[0193] 8A and 8B show difference plots of the HRM curves of FIGS. 7A and 7B, respectively.

[0194] From this experiment, it can be concluded that the application of temperature shift reduces the differences between healthy samples from different patients, which creates the possibility of using one universal reference sample instead of paired normal and tumor samples for each patient.

[0195] Example 6: Asymmetric PCR creates more single-stranded amplicons. NR22 assay using asymmetric PCR with primers hybridizing upstream and downstream of the region containing the mononucleotide repeat. The assay is for detecting mutant sequences in NR22 microsatellites. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 142-172 of SEQ ID NO:4, has an overhang at its 3' end, and a helper sequence at its 5' end (resulting in an increase in Tm of 14.2°C), which hybridizes upstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end, and a hydrophobic nucleotide within 10 nucleotides from the 3' end.

[0196] Blood purified normal tissue DNA of 1-2 ng / μL was tested. In the figure, the real-time PCR curves are seen. The symmetric PCR curve stops increasing in RFU after 45 cycles, while for the asymmetric PCR, the amplification continues after the exponential growth phase with linear amplification.

[0197] This experiment confirms that asymmetric PCR creates more single DNA strands that contain sequences to which the hybridization sequence of the reporter oligonucleotide can bind.

[0198] Example 7: Asymmetric PCR produces higher signal-to-noise ratios and sharper melting curves. The assay was performed as described in Example 6. Normalized HRM curves were obtained and are shown in FIG.

[0199] From this experiment it can be concluded that asymmetric PCR creates a higher signal to noise ratio and produces a "sharper" melting profile.

[0200] Example 8: Asymmetric PCR makes it easier to distinguish between MSS and MSI patients. The NR22 assay was performed as described in Example 6. 1-2 ng / μL of FFPE purified normal and tumor tissue DNA from microsatellite instability patients was tested.

[0201] Figure 11 shows the HRM curves obtained after applying standard normalization using asymmetric PCR (Figure 11A) or symmetric PCR (Figure 11B). The corresponding difference curves are shown in Figures 12A and 12B, respectively.

[0202] From this experiment, it can be concluded that amplification using asymmetric PCR creates a greater difference between normal and tumor tissue curves for microsatellite instability patients compared to symmetric PCR, thus making it easier to distinguish between MSS and MSI patients.

[0203] Example 9: Single-end overhangs in reporter oligonucleotides can increase the melting temperature. An NR22 assay using asymmetric PCR with primers that hybridize upstream and downstream of a region containing a mononucleotide repeat, the assay is for detecting variant sequences in the NR22 microsatellite.

[0204] Two different reporter oligonucleotides were used in this experiment. The first reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end. The first reporter oligonucleotide hybridizes to positions 148-177 of SEQ ID NO:4. The hybridization sequence also contains a 4 nucleotide long helper sequence at the 5' end (resulting in an increase in Tm of 7.5°C) and a 5 nucleotide long helper sequence at the 3' end (resulting in an increase in Tm of 3.6°C), which are complementary to regions of NR22 immediately upstream and downstream of the repeat. The second reporter oligonucleotide carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end as well as an internal quencher. The second reporter oligonucleotide hybridizes to positions 143-172 of SEQ ID NO:4 and has an overhang at its 3' end and only a 9 nucleotide long helper sequence at its 5' end (resulting in an increase in Tm of 14.2°C), which hybridizes upstream of the mononucleotide repeat. In addition, both reporter oligonucleotides contain a hydrophobic nucleotide within 10 nucleotides from the 5' end and a hydrophobic nucleotide within 10 nucleotides from the 3' end. Blood purified normal tissue DNA at 1-2 ng / μL was tested.

[0205] Results for assays using the first reporter oligonucleotide are shown as dashed lines in Figure 13; results using the second reporter oligonucleotide are shown as solid lines. Melting curves are shown in Figure 13A and derivative curves in Figure 13B. Bilinear normalization and temperature shifts were not applied.

[0206] It is confirmed that the melting temperature for the second reporter oligonucleotide is higher than that for the first reporter oligonucleotide. The lower background fluorescence for the second reporter oligonucleotide may be due to the presence of an additional internal quencher.

[0207] This experiment shows that having one longer helper sequence at one end instead of two smaller helper sequences at both ends can increase the melting temperature of the probe, which can be advantageous when interrogating mononucleotide repeat microsatellites.

[0208] Example 10: Dual quenching of reporter oligonucleotides increases the signal-to-noise ratio. BAT26 assay using asymmetric PCR with primers hybridizing upstream and downstream of a region containing a mononucleotide repeat. The assay is to detect mutant sequences in the BAT26 microsatellite. 1-2 ng / μL of blood purified normal tissue DNA was tested. A: HRM curve with standard normalization applied. In this experiment, two reporter oligonucleotides were used, both carrying a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end. As an example of a reporter oligonucleotide that is only single quenched, but not a DQ probe, the BAT26 reporter oligonucleotide FAM5'-CZCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAZC-3'BHQ-1 (SEQ ID NO: 7) was used. The BAT26 reporter oligonucleotide FAM5'-CZCCTTTTTTTTTTXTTTTTTTTTTTTTTTTTTAZC-3'BHQ-1 (SEQ ID NO:8) was used to illustrate the doubly quenched, DQ, reporter oligonucleotide; X indicates an internal quencher.

[0209] The reporter oligonucleotide hybridizes to positions 220-251 of SEQ ID NO:2, has an overhang at its 3' end and helper sequences at both its 5' end (resulting in an increase in Tm of 5.2° C.) and its 3' end (resulting in an increase in Tm of 1.9° C.), which hybridize upstream and downstream of the mononucleotide repeat. In addition, it contains a hydrophobic nucleotide within 10 nucleotides from the 5' end and a hydrophobic nucleotide within 10 nucleotides from the 3' end.

[0210] The HRM curve and the negative first derivative curve are shown in Figure 14A and B, respectively. It is confirmed that the background fluorescence of the single-quenched reporter oligonucleotide (but not the DQ probe) is higher than that of the double-quenched reporter oligonucleotide (the DQ probe). From this experiment, it is concluded that the DQ reporter oligonucleotide reduces the fluorescence noise and makes the melting sharper compared to the single-quenched reporter oligonucleotide. The use of the double-quenched reporter oligonucleotide can therefore facilitate the discrimination between normal and tumor samples.

[0211] Example 11: Additional nucleotide repeats in the hybridization sequence of the reporter oligonucleotide allow longer microsatellites to be detected. The NR21 assay was carried out using asymmetric PCR. The assay is to detect mutant sequences in the NR21 microsatellite. The reporter oligonucleotide used in this example carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The reporter oligonucleotide hybridizes to positions 189-214 of SEQ ID NO:2, has an overhang at its 3' end, and a helper sequence at its 5' end (resulting in an increase in Tm of 15.2°C), which hybridizes downstream of the mononucleotide repeat. In addition, the reporter oligonucleotide contains two hydrophobic nucleotides within 5 nucleotides from the 5' end and one hydrophobic nucleotide within 5 nucleotides from the 3' end. The hybridization sequence of the reporter oligonucleotide contains a nucleotide repeat of 22 T; the NR21 microsatellite consists of 21 repeats in most normal cells. The reporter oligonucleotide therefore has one additional T compared to the reference sequence.

[0212] Reporter oligonucleotides were tested against artificial targets of 21, 22 or 23 adenine repeats.

[0213] The HRM curves after applying bilinear normalization but without applying temperature shift are shown in Figure 15. From this experiment, it can be concluded that adding one extra repeat in the hybridization sequence of the reporter oligonucleotide allows the detection of microsatellites longer than the reference 21 repeats.

[0214] Example 12: A single point mutation alters the melting temperature by several degrees. KIT exon 13 assay for detecting mutant sequences in KIT exon 13. PCR was performed as an asymmetric PCR. The reporter oligonucleotide carried a FAM fluorophore at its 5' end and a BHQ1 quencher at its 3' end. 1 ng / ul of FFPE purified tissue DNA from wild type samples and FFPE purified tumor tissue from patient samples were examined. The initial normalization interval was 70-71°C and the final normalization interval was 80-81°C. The results are shown in Figure 16. Wild type tissue had a T of 79.1°C. m The tumor tissue was then divided into two groups: m ; giving 75.7°C and 79.1°C.

[0215] From this experiment it can be concluded that the melting temperature of the probe is altered by up to 3.4° C. and the shape of the melting curve changes significantly in case of single point mutations, thus allowing differentiation of healthy and tumor samples.

[0216] Example 13: Stronger single-end helper sequences increase differentiation and facilitate differentiation of mutants and wild type. The NR24 assay uses asymmetric PCR with primers that hybridize upstream and downstream of the region containing the mononucleotide repeat. The assay is to detect mutant sequences in the NR24 microsatellite.

[0217] Two different reporter oligonucleotides were used in this experiment. The first reporter oligonucleotide carried a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end as well as an internal quencher. The first reporter oligonucleotide hybridizes to positions 159-187 of SEQ ID NO:5, has an overhang (2 nucleotides) at its 3' end, and a 5 nucleotide long helper sequence at its 5' end (resulting in an increase in Tm of 10.5°C), which hybridizes upstream of the mononucleotide repeat. In addition, it contains two hydrophobic nucleotides within 10 nucleotides from the 5' end and a hydrophobic nucleotide within 10 nucleotides from the 3' end. The second reporter is described in Example 4. FFPE purified normal and tumor tissue DNA from microsatellite instability patients at 1-2 ng / μL was tested.

[0218] Initial normalization was performed in the region 40.5-41.5°C, final normalization in the region 66-67°C. Bilinear normalization and a temperature shift of 0.05 RFU were applied. The results are shown in Figures 17 and 18; compare Figures 17A and 18A (results with the first reporter oligonucleotide) and Figures 17B and 18B (results with the second reporter oligonucleotide).

[0219] When hybridized, the second helper sequence results in a higher increase in Tm (14.8° C., ie, 4.3° C. higher) than the first helper sequence, which serves to distinguish the two melting profiles.

[0220] The stronger helper sequence is the maxD T The first reporter oligonucleotide with a weak helper sequence is confirmed to increase the maxD of about 0.03 RFU between normal and tumor tissues. T This is below the threshold set for the NR24 marker, so the mutant is misclassified as wild type (Figure 18A). The second reporter oligonucleotide, which has a stronger helper sequence, yielded a maxD of approximately 0.17. T, and thus the sample is correctly classified as mutant (Figure 18B).

[0221] From this experiment it can be concluded that stronger helper sequences can increase the differentiation between wild type and mutants, which may be advantageous when investigating mononucleotide repeat microsatellites.

[0222] array [Table 1] TIFF0007681520000002.tif204162

[0223] References Boland et al. (1998) "A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer," Cancer Res 58:5248-5257 Rodriguez-Bigas et al. (1997) "A National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines," J Natl Cancer Inst 89:1758-1762 Hunter and Sanders (1990) J. Am Chem. Soc. 112: 5525-5534 WO 2017 / 045689 WO 03 / 052132

[0224] Terms 1. A method for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), said target nucleic acid sequence consisting of a variant sequence or a reference sequence, said method comprising the following steps: a) providing a first sample containing nucleic acid suspected of containing the mutant sequence; b) preparing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence; e) amplifying a target nucleic acid sequence in the presence of the first sample, the first primer, and the second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a mutant sequence; and amplifying a target nucleic acid sequence in the presence of the second sample, the first primer, and the second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a high resolution melting (HRM) analysis of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of the second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, the second HRM profile being a reference profile characterized by a reference melting curve, each amplicon comprising a first strand and a second strand, the HRM analysis comprising hybridization of a reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by a fluorophore, and obtaining the first and second melting curves; (Reporter oligonucleotides are sequences of 10-50 nucleotides with 2-10 hydrophobic nucleotides inserted therein. the reporter oligonucleotide comprises a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end; and The reporter oligonucleotide comprises a hybridization sequence H, at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; and g) comparing the first HRM profile with a reference HRM profile, wherein a difference between the first HRM profile and the reference HRM profile indicates that the first sample contains a variant sequence. A method comprising:

[0225] 2. The method of claim 1, wherein the NOI comprises repeats and the hybridization sequence of the reporter oligonucleotide consists of the repeat sequence and a helper sequence at its 5' end and / or its 3' end, said helper sequence being capable of hybridizing to the first or second strand of the first and second amplicons when it does not comprise repeats and the hybridization sequence hybridizes thereto, preferably the reporter oligonucleotide consists of the repeat sequence and only one helper sequence at its 5' end or its 3' end.

[0226] 3. The method of claim 2, wherein the helper sequence comprises or consists of 1 to 20 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, preferably wherein the helper sequence comprises at least one hydrophobic oligonucleotide as defined in clause 1.

[0227] 4. The method of any one of clauses 1 to 3, wherein at least one hydrophobic nucleotide, such as 1, 2 or 3 hydrophobic nucleotides as defined in clause 1, is inserted within 1 to 10 nucleotides from the 3' end, such as within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 3' end of the reporter oligonucleotide.

[0228] 5. The method of any one of clauses 1 to 4, wherein at least one hydrophobic nucleotide, such as 1, 2 or 3 hydrophobic nucleotides as defined in clause 1, is inserted within 1 to 10 nucleotides from the 5' end, such as within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 5' end of the reporter oligonucleotide.

[0229] 6. The method of any one of clauses 1 to 5, wherein the plurality of target nucleic acid sequences consists of BAT25 and BAT26, preferably the plurality consists of BAT25 as set forth in SEQ ID NO:1 and BAT26 as set forth in SEQ ID NO:2.

[0230] 7. The method of any one of clauses 1 to 6, wherein the target nucleic acid sequence is one or more nucleic acid sequences including one or more of BAT25, BAT26, NR21, NR22, NR24 and MONO27, preferably BAT25 as set forth in SEQ ID NO:1, BAT26 as set forth in SEQ ID NO:2, NR21 as set forth in SEQ ID NO:3, NR22 as set forth in SEQ ID NO:4, NR24 as set forth in SEQ ID NO:5 and MONO27 as set forth in SEQ ID NO:6.

[0231] 8. The method of any one of clauses 1 to 7, wherein the target nucleic acid is a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR21, NR22 and NR24, preferably the plurality consists of BAT25 as set forth in SEQ ID NO:1, BAT26 as set forth in SEQ ID NO:2, NR21 as set forth in SEQ ID NO:3, NR22 as set forth in SEQ ID NO:4, and NR24 as set forth in SEQ ID NO:5.

[0232] 9. The method of any one of clauses 1 to 8, wherein the target nucleic acid is a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR22, NR24 and MONO27, preferably the plurality consists of BAT25 as set forth in SEQ ID NO:1, BAT26 as set forth in SEQ ID NO:2, NR22 as set forth in SEQ ID NO:4, NR24 as set forth in SEQ ID NO:5 and MONO27 as set forth in SEQ ID NO:6.

[0233] 10. The method of any one of clauses 1 to 9, wherein the target nucleic acids are a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR21, NR22, NR24 and MONO27, preferably the plurality of target nucleic acid sequences consisting of BAT25 as set forth in SEQ ID NO:1, BAT26 as set forth in SEQ ID NO:2, NR21 as set forth in SEQ ID NO:3, NR22 as set forth in SEQ ID NO:4, NR24 as set forth in SEQ ID NO:5 and MONO27 as set forth in SEQ ID NO:6.

[0234] 11. The method of any one of clauses 1 to 10, wherein the amplification in step e) is carried out by polymerase chain reaction (PCR), preferably by asymmetric PCR, in which the first and second primers are provided in different amounts, thereby directing the PCR to amplify more of the second strand of each amplicon than the first strand of each amplicon.

[0235] 12. The method of any one of clauses 1 to 11, wherein the first sample is isolated from an individual suffering from or suspected of suffering from a disease such as cancer, preferably wherein the cancer is hereditary non-polyposis colorectal cancer.

[0236] 13. The method of any one of clauses 1 to 12, wherein the NOI is a microsatellite.

[0237] 14. The method of any one of clauses 1 to 13, wherein the reference sequence comprises a microsatellite sequence of M tandem repeats having a total length of n nucleotides, and the variant sequence has M' tandem repeats having a total length of n' nucleotides, where M and M' are different integers.

[0238] 15. The method of any one of clauses 1 to 14, wherein the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1, preferably n"≧n+1 or n"≧n+2.

[0239] 16. The method of any one of clauses 1 to 15, wherein the reporter oligonucleotide comprises a second quencher located at a non-terminal position of the reporter oligonucleotide.

[0240] 17. The method of any one of clauses 1 to 16, wherein the first sample is a sample of tissue containing or suspected of containing cells having a mutation characteristic of said disease.

[0241] 18. The method of any one of clauses 1 to 17, wherein the reference sample is isolated from the same individual as the first sample, optionally from non-diseased tissue, or the reference sample is isolated from a healthy individual.

[0242] 19. The method of any one of clauses 1 to 18, wherein step f) comprises transforming the first and second HRM melting curves to obtain negative first derivatives of the first and second melting curves, and the difference between the first HRM profile and the reference HRM profile is the difference between the negative first derivatives of the first and second melting curves.

[0243] 20. The method of any one of conditions 1 to 19, wherein the fluorescence of the first melting curve and the fluorescence of the second melting curve are normalized and the difference between the HRM profiles is the difference between the fluorescence of the first and second melting curves as a function of temperature.

[0244] 21. The method of any one of clauses 1 to 20, wherein the difference between the first HRM profile and the reference HRM profile is measured as the absolute maximum difference in relative fluorescence units within the boundaries of the upper and lower normalized regions between the first and second HRM curves.

[0245] 22. The method of any one of clauses 1 to 21, wherein the HRM analysis comprises a step of bilinear normalization of the first and second melting curves.

[0246] 23. The method of any one of clauses 1 to 22, wherein the HRM analysis comprises applying a temperature adjustment in relative fluorescence units (RFU) to the first and second melting curves and / or to the negative first derivatives of the first and second melting curves.

[0247] 24. The method of any one of clauses 1 to 23, wherein the second primer comprises a sequence of at least 15 nucleotides that is complementary to a contiguous sequence of the target nucleic acid sequence.

[0248] 25. The method of any one of clauses 1 to 24, wherein the variant sequence comprises or consists of an insertion of one or more nucleotides compared to the reference sequence.

[0249] 26. The method of any one of clauses 1 to 25, wherein the variant sequence comprises or consists of a deletion of one or more nucleotides compared to the reference sequence.

[0250] 27. The reporter oligonucleotide has the general formula: 5'-(N) a -Z-(N) d- Z-(N) e -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and the total number of nucleotides or nucleotide analogs is at least 10; and a and b are each an integer ranging from 0 to 4; and d and e are each an integer ranging from 1 to 19; and a+b+d+e is at least 10) having; (N) a -(N) d -(N) e -(N) b is identical to the reference sequence, 27. The method according to any one of clauses 1 to 26.

[0251] 28. The reporter oligonucleotide has the following general structure: 5'-(N) a -Z-(N) f- Z-(N) g -Z-(N) h -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and a and b are each an integer ranging from 0 to 4; and f, g and h are individually integers ranging from 1 to 18; and a+b+f+g+h is at least 10 and at most 50; and (N) a -(N) f -(N) g -(N) h -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) i -Z-(N) j -Z-(N) k -Z-(N) l -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and a and b are each an integer ranging from 0 to 4; and i, j, k and l are integers ranging from 1 to 17; and a+b+i+j+k+l are individually at least 10 and at most 50; and (N) a -(N) i -(N) j -(N) k -(N) l -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having; or The general structure 5'-(N) a -Z-(N) m -Z-(N) n -Z-(N) o -Z-(N) p -Z-(N) q -Z-(N) b -3' (In the formula, N is any nucleotide or nucleotide analog; and Z is a hydrophobic nucleotide as defined in clause 1; and a and b are each an integer ranging from 0 to 4; and m, n, o, p and q are individually integers ranging from 1 to 16; and a+b+m+n+o+p+q is at least 10 and at most 50; and (N) a -(N) m -(N) n -(N) o -(N) p -Z-(N) q -(N) b is identical to a stretch of the target nucleic acid sequence that includes the reference sequence) having 28. The method according to any one of clauses 1 to 27.

[0252] 29. The method of any one of clauses 1 to 28, wherein at least one intercalator, Q, is selected from the group consisting of polyaromates and heteropolyaromates, optionally substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carboalkoyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, and amido.

[0253] 30. The method according to any one of clauses 1 to 29, wherein the intercalator is selected from the group consisting of benzene, pentalene, indene, naphthalene, azulene, as-indacene, s-indacene, biphenylene, acenaphthylene, phenalene, heptalene, phenanthrene, fluoranthene, phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, fluorene, picene, chrysene, naphthacene, acridone, benzanthracene, stilbene, oxalo-pyridocarbazole, azidobenzene, porphyrin and psoralen and derivatives thereof.

[0254] 31. The method according to any one of clauses 1 to 30, wherein at least one, for example all, of the backbone monomer unit(s) X is a phosphoramidite.

[0255] 32. The method according to any one of clauses 1 to 31, wherein at least one linker, Y, comprises a chain of x atoms selected from the group consisting of C, O, S, N and P, optionally wherein the chain is substituted with one or more selected from the group consisting of C, H, O, S, N and P.

[0256] 33. The method according to any one of clauses 1 to 32, wherein the target nucleic acid sequence is a plurality of target nucleic acid sequences.

[0257] 34. A kit of parts for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and containing the nucleotide(s) of interest (NOI), wherein the target nucleic acid sequence consists of a variant sequence or a reference sequence, and the kit of parts comprises: a) a reporter oligonucleotide comprising a first fluorophore, preferably at or within 4 nucleotides from its 5'-end, and a first quencher, preferably at or within 4 nucleotides from its 3'-end; (The reporter oligonucleotide is a sequence of 10-50 nucleotides having 2-10 hydrophobic nucleotides inserted therein, the reporter oligonucleotide including a hybridization sequence H), (at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid; and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid; Including, The above is a kit of parts.

[0258] 35. The kit of clause 34, wherein the reporter oligonucleotide and the first and second primers are as defined in any one of clauses 1 to 34.

[0259] 36. A reporter oligonucleotide capable of hybridizing to one strand of a target nucleic acid, comprising a first fluorophore, preferably at or within 4 nucleotides from its 5' end, and a first quencher, preferably at or within 4 nucleotides from its 3' end, wherein the reporter oligonucleotide is a sequence of 10-50 nucleotides with 2-10 hydrophobic nucleotides inserted therein, wherein the reporter oligonucleotide comprises a hybridization sequence H; at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure XYQ (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding; and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator; and the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid; Reporter oligonucleotides.

[0260] 37. A reporter oligonucleotide according to clause 36, wherein the NOI comprises repeats and the hybridisation sequence of the reporter oligonucleotide consists of the repeat sequence and a helper sequence at its 5' end and / or its 3' end, the helper sequence not comprising repeats and capable of hybridising to the second strand of the target nucleic acid when the hybridisation sequence hybridises to the target nucleic acid, preferably the reporter oligonucleotide consists of the repeat sequence and only one helper sequence at its 5' end or its 3' end.

[0261] 38. The reporter oligonucleotide according to any one of clauses 36 to 37, wherein the helper sequence comprises or consists of 1 to 20 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, preferably wherein the helper sequence comprises at least one hydrophobic oligonucleotide as defined in clause 1.

[0262] 39. The reporter oligonucleotide of any one of clauses 36 to 38, wherein the reference sequence comprises a microsatellite sequence of M tandem repeats having a total length of n nucleotides, and the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1, preferably n"≧n+1 or n"≧n+2.

[0263] 40. The reporter oligonucleotide of any one of clauses 36 to 39, wherein the reporter oligonucleotide comprises a second quencher located at a non-terminal position of the reporter oligonucleotide.

[0264] 41. A method for predicting the efficacy of treating a clinical condition in an individual in need thereof with a given drug, wherein the efficacy of treating said clinical condition with said drug is related to the presence of a variant sequence, said method comprising the steps of: a. Preparing a sample from said individual b. carrying out the method according to any one of clauses 1 to 33 to determine the presence of said variant sequence. Includes; The presence of said variant sequence indicates whether said agent will be effective in treating said clinical condition in said individual.

[0265] 42. A method for predicting the presence of a clinical condition in an individual in need thereof, wherein the clinical condition is associated with the presence of a target nucleic acid sequence comprising a mutant sequence, the method comprising the steps of: a. Preparing a sample from said individual b. carrying out the method of any one of clauses 1 to 33 to determine the presence of the variant sequence. Includes; The presence of said variant sequence is indicative of said individual suffering from said clinical condition.

[0266] 43. The method according to any one of clauses 41 to 42, wherein the clinical condition is cancer, preferably hereditary non-polyposis colorectal cancer.

[0267] 44. The method of any one of clauses 41 to 43, further comprising administering to said individual an effective amount of a therapeutic agent.

Claims

1. 1. A method for detecting the presence of a variant sequence in a target nucleic acid sequence which is comprised of two strands and contains one or more nucleotides of interest (NOIs), comprising: The NOI is a microsatellite comprising a microsatellite sequence of M tandem repeats having a total length of n nucleotides, the variant sequence has M' tandem repeats having a total length of n' nucleotides, M and M' being different integers, n being 15 or greater, the target nucleic acid sequence consists of a variant sequence or a reference sequence, the method comprising the steps of: a) providing a first sample containing nucleic acid suspected of containing said mutant sequence; b) providing a second sample comprising a nucleic acid comprising the reference sequence, the second sample being a reference sample; c) providing a reporter oligonucleotide; d) providing a set of primers consisting of a first primer and a second primer, the set of primers together being capable of amplifying a target nucleic acid sequence comprising the NOI; e) amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby obtaining a first amplicon comprising a nucleic acid suspected of comprising a variant sequence; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby obtaining a second amplicon comprising a reference sequence, wherein the second amplicon is a reference amplicon; f) performing a melting analysis of the first amplicon, thereby obtaining a first profile characterized by a first melting curve, and performing a melting analysis of the second amplicon, thereby obtaining a second profile characterized by a second melting curve, the second profile being a reference profile characterized by a reference melting curve; Each amplicon comprises a first strand and a second strand, The melting analysis includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by the fluorophore, and obtaining a first and a second melting curve; The reporter oligonucleotide is a sequence ranging from 15 to 50 nucleotides, having hydrophobic nucleotides inserted therein ranging from 2 to 10, the reporter oligonucleotide comprises a first fluorophore and a first quencher; The reporter oligonucleotide comprises a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid sequence, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid sequence; the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1 or n"≧n+2; the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or at its 3' end, said helper sequence being free of the repeat sequence and capable of hybridizing to the first and second amplicons when the hybridization sequence is hybridized thereto; and g) comparing the first profile to a reference profile, wherein a difference between the first profile and the reference profile indicates that the first sample contains a mutant sequence. A method comprising:

2. Step g) comprises the steps of: i) aligning the first and second melting curves at a given fluorescence intensity along the temperature axis, thereby negating the difference in melting temperature between the first and second melting curves; ii) determining the difference in the signal emitted by the fluorophore between the first and second melting curves, the difference being a numerical difference; and iii) comparing the difference determined in step ii) to a threshold value, wherein a difference greater than the threshold value indicates that the first sample contains the variant sequence and a difference less than the threshold value indicates that the first sample contains the reference sequence.

2. The method of claim 1, comprising or consisting of:

3. The method of claim 1 , wherein the reference sequence has a length of 15 nucleotides or more.

4. 4. The method of claim 1, wherein the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or its 3' end, or the reporter oligonucleotide consists of a repeat sequence and two helper sequences.

5. at least one said hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and The hydrophobic nucleotide has the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator.

5. The method according to claim 1 , further comprising:

6. The method according to any one of claims 1 to 5, wherein the helper sequence comprises or consists of 1 to 20 nucleotides.

7. At least one of the 1 to 20 nucleotides is a hydrophobic nucleotide, the hydrophobic nucleotide having the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator.

7. The method of claim 6, comprising:

8. The reporter oligonucleotide comprises at least one hydrophobic nucleotide within 1 to 10 nucleotides from the 3' end, the hydrophobic nucleotide having the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator.

6. The method according to claim 1 , comprising:

9. The reporter oligonucleotide comprises at least one hydrophobic nucleotide within 1 to 10 nucleotides from the 5' end, the hydrophobic nucleotide having the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator.

6. The method according to claim 1 , comprising:

10. 10. The method of any one of claims 1 to 9, wherein the amplification in step e) is carried out by polymerase chain reaction (PCR), and the first and second primers are provided in different amounts, thereby directing the PCR to amplify one strand of each amplicon more than the other strand of each amplicon.

11. 11. The method of any one of claims 1 to 10, wherein the first sample is isolated from an individual suffering from or suspected of suffering from a disease.

12. 12. The method of any one of claims 1 to 11, wherein the first sample is a sample of tissue containing or suspected of containing cells with mutations characteristic of a disease or disorder.

13. 1. A kit for detecting the presence of a variant sequence in a target nucleic acid sequence, the target nucleic acid sequence consisting of two strands and containing at least one nucleotide of interest (NOI), comprising: wherein the NOI is a microsatellite comprising a microsatellite sequence of M tandem repeats having a total length of n nucleotides, the variant sequence has M' tandem repeats having a total length of n' nucleotides, M and M' being different integers, n being 15 or greater, and the target nucleic acid sequence consists of the variant sequence or the reference sequence, and the kit comprises: a) a reporter oligonucleotide comprising a first fluorophore and a first quencher, the reporter nucleotide is a sequence ranging from 15 to 50 nucleotides, into which are inserted hydrophobic nucleotides ranging from 2 to 10; The reporter oligonucleotide comprises a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of the first strand of the target nucleic acid, and the hybridization sequence is complementary to a contiguous stretch of the sequence of the second strand of the target nucleic acid; the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1 or n"≧n+2, and n" is 16 or greater; the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or at its 3' end, said helper sequence being free of the repeat sequence and capable of hybridizing to the first and second amplicons when the hybridization sequence is hybridized thereto; a reporter oligonucleotide, and b) a set of primers consisting of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence; Including the kit.

14. 14. The kit of claim 13, wherein the reporter oligonucleotide, the first primer and the second primer are as defined in any one of claims 1 to 6 or 10 to 12.

15. 15. A kit according to any one of claims 13 to 14, wherein the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or at its 3' end, or wherein the reporter oligonucleotide consists of a repeat sequence and two helper sequences.

16. at least one said hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or at least one hydrophobic nucleotide is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator. The kit according to any one of claims 13 to 14, comprising:

17. a reporter oligonucleotide which is capable of hybridizing to one strand of a target nucleic acid which consists of two strands and contains at least one nucleotide of interest (NOI), the NOI is a microsatellite comprising a microsatellite sequence of M tandem repeats having a total length of n nucleotides, the variant sequence has M' tandem repeats having a total length of n' nucleotides, M and M' being different integers, n being 15 or greater, said reporter oligonucleotide comprising a first fluorophore and a first quencher, the reporter oligonucleotide being a sequence in the range of 15-50 nucleotides having inserted therein a range of 2-10 hydrophobic nucleotides, and the reporter oligonucleotide comprising a hybridization sequence H, the hybridization sequence is identical to a contiguous stretch of the sequence of a first strand of the target nucleic acid and the hybridization sequence is complementary to a contiguous stretch of the sequence of a second strand of the target nucleic acid, the length of the hybridization sequence H of the reporter oligonucleotide is n", where n"≧n+1 or n"≧n+2, and n" is 16 or more; the hybridization sequence of the reporter oligonucleotide comprises or consists of a repeat sequence and at least one helper sequence at its 5' end and / or at its 3' end, said helper sequence being free of repeat sequences and capable of hybridizing to the second strands of the first and second amplicons when the hybridization sequence is hybridized thereto; Reporter oligonucleotides.

18. 18. The reporter oligonucleotide of claim 17, which consists of a repeat sequence and only one helper sequence at its 5' end or its 3' end, or which consists of a repeat sequence and two helper sequences.

19. at least one said hydrophobic nucleotide is located at or within 10 nucleotides from the 5' end of the reporter oligonucleotide; and / or At least one of the hydrophobic nucleotides is located at or within 10 nucleotides from the 3' end of the reporter oligonucleotide; and Hydrophobic nucleotides have the structure X-Y-Q (In the formula, X is a nucleotide or nucleotide analog or backbone monomer unit that can be incorporated into the backbone of a nucleic acid or nucleic acid analog; Q is an intercalator that is not involved in Watson-Crick hydrogen bonding, and Y is a linker moiety connecting the nucleotide or nucleotide analog or backbone monomer unit and the intercalator.

19. The reporter oligonucleotide of any one of claims 17 to 18, having the following structure:

20. The reporter oligonucleotide of any one of claims 17 to 18, wherein the helper sequence comprises or consists of 1 to 20 nucleotides.

21. 1. A method for predicting the efficacy of treating a clinical condition in an individual in need thereof with a given drug, the efficacy of treating said clinical condition with said drug being related to the presence of a variant sequence, said method comprising the steps of: a. Providing a sample from said individual b) carrying out the method of any one of claims 1 to 11 to determine the presence of said mutant sequence. Including; The presence of said variant sequence indicates whether said agent will be effective in treating said clinical condition in said individual.

22. 1. A method for predicting the presence of a clinical condition in an individual in need thereof, wherein said clinical condition is associated with the presence of a target nucleic acid sequence comprising a variant sequence, said method comprising the steps of: a. Providing a sample from said individual b. carrying out the method of any one of claims 1 to 12 to determine the presence of a mutant sequence. Including; wherein the presence of the variant sequence is indicative that said individual suffers from said clinical condition.

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