Methods for detecting fusion genes

Abstract

Providing a method for detecting fusion genes. [Solution] The present invention provides a method for detecting a fusion gene derived from gene I and gene II, comprising the steps of: performing an amplification reaction on a nucleic acid sample containing a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II; performing a ligation reaction on the nucleic acid sample on which the amplification reaction has been performed in the presence of a crosslinking probe; and analyzing the nucleic acid sample on which the ligation reaction has been performed.

Description

Technical Field

[0001] The present invention broadly relates to a method for detecting a fusion gene and the like.

Background Art

[0002] A fusion gene generated by the linkage of two genes due to chromosomal translocation, inversion, etc. can cause cancer. Various molecular target drugs targeting these have been developed and marketed. Since there are many types of fusion genes depending on the combination of genes to be fused and the chromosomal breakpoints, in the Fluorescent in situ hybridization (FISH) method, qPCR method, etc. known so far as methods for detecting fusion genes, detection probes corresponding to each fusion gene were required to detect each fusion gene.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The problem to be solved by the present invention is to provide a novel method for detecting a fusion gene. Another problem to be solved by the present invention is also to provide a novel method capable of easily detecting a fusion gene without preparing a detection probe for each fusion gene.

Means for Solving the Problems

[0004] As a result of intensive studies by the present inventors, it has been found that a fusion gene can be detected by using a circular DNA oligo containing the sequence of the fusion part of the fusion gene. Also, by including a predetermined sequence in the circular DNA oligo, it has been found that a fusion gene can be easily detected without preparing a detection probe for each fusion gene.

[0005] That is, the present application includes the following inventions. [1] A method for producing a circular DNA oligo containing the sequence of the fusion portion of a fusion gene derived from gene I and gene II, A step of obtaining an amplicon by amplifying a region containing the sequence of the fusion portion of a fusion gene in the presence of a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II. A process in which an amplicon hybridizes with a crosslinking probe, and A method comprising the step of joining the 5' and 3' ends of an amplicon hybridized to a crosslinking probe in the presence of a ligase. [2] A circular DNA oligo further contains a predetermined sequence, The method according to [1], wherein a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II each further comprises a single-stranded nucleic acid region at their 5' ends derived from a sequence obtained by splitting a predetermined sequence into two at any position. [3] The method according to [2], wherein the crosslinking probe has an arrangement corresponding to part or all of a predetermined arrangement. [4] The method according to [2] or [3], wherein the specified sequence is not a human-derived sequence. [5] The method according to any one of [2] to [4], wherein the length of a given sequence is 10 to 400 nucleotides. [6] The method described in any one of [1] to [5], wherein the step of obtaining the amplicon is performed by multiplex PCR. [7] The method described in any one of the following [1] to [6], wherein the fusion gene is a cancer-causing gene. [8] A method for detecting a fusion gene derived from gene I and gene II, A step of performing an amplification reaction on a nucleic acid sample containing a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II. A step of performing a ligation reaction on a nucleic acid sample that has undergone an amplification reaction in the presence of a crosslinking probe, and A method comprising the step of analyzing a nucleic acid sample on which a ligation reaction has been performed. [9] The method according to [8], wherein the step of carrying out the amplification reaction is the step of obtaining an amplicon containing the sequence of the fusion portion of the fusion gene.

[10] The method according to [9], wherein the step of carrying out a ligation reaction in the presence of a crosslinking probe is to join the 5' and 3' ends of an amplicon hybridized to the crosslinking probe in the presence of a ligase to obtain a circular DNA oligo containing the sequence of the fusion portion of the fusion gene.

[11] The process of analyzing nucleic acid samples after ligation reactions is The method according to

[10] , which involves analyzing the amplification reaction of a region containing a ligase-mediated junction in a circular DNA oligo.

[12] The method according to any one of [8] to

[11] , wherein a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II each further comprises a single-stranded nucleic acid region at their 5' ends derived from a sequence obtained by splitting a predetermined sequence into two at any position.

[13] The method according to

[12] , wherein the crosslinking probe has an arrangement corresponding to part or all of a predetermined arrangement.

[14] The method according to

[12] or

[13] , wherein the sequence of the region including the junction corresponds to a sequence that corresponds to a part or all of a predetermined sequence.

[15] The method according to any one of

[12] to

[14] , wherein the given sequence is not a human-derived sequence.

[16] A kit for detecting fusion genes derived from gene I and gene II, A primer containing a portion of the sequence of gene I, and a primer containing a portion of the sequence of gene II, and A kit including a crosslinking probe.

[17] The kit further comprises a primer having a sequence of a portion of a given sequence, A primer containing a partial sequence of gene I and a primer containing a partial sequence of gene II each further contain a single-stranded nucleic acid region derived from a sequence obtained by splitting a predetermined sequence into two at the 5'-end. The kit according to

[16] , wherein the bridging probe has a sequence corresponding to a part or all of a predetermined sequence. [Advantages of the Invention]

[0006] According to the present invention, it becomes possible to provide a novel method for detecting a fusion gene. Furthermore, according to the present invention, it also becomes possible to provide a novel method that can simply detect a fusion gene without preparing a detection probe for each fusion gene. [Brief Description of the Drawings]

[0007] [Figure 1] The results of performing quantitative real-time PCR using a fully circularized RT-PCR amplicon having a region containing the chromosomal breakpoint of COSF571 or COSF1326 are shown. [Modes for Carrying Out the Invention]

[0008] Hereinafter, embodiments of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention is not construed as being limited to the following embodiments.

[0009] In a first embodiment, a method for detecting a fusion gene derived from gene I and gene II, comprising: performing an amplification reaction on a nucleic acid sample containing a primer containing a partial sequence of gene I and a primer containing a partial sequence of gene II; performing a ligation reaction on the nucleic acid sample on which the amplification reaction has been performed in the presence of a bridging probe; and analyzing the nucleic acid sample on which the ligation reaction has been performed.

[0010] As used herein, the term "fusion gene" refers to a gene generated by the ligation of two types of genes due to chromosomal translocation, inversion, or the like. The fusion gene detected by the method of the present embodiment is preferably a causative gene of cancer. In the present embodiment, genes I and II that form a fusion gene may be any combination of different genes as long as they are different from each other. The fusion gene derived from genes I and II includes any region of gene I and any region of gene II. The combination of genes I and II may be appropriately determined according to the type of fusion gene that needs to be detected.

[0011] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a chimeric nucleic acid thereof, and may be an artificially synthesized nucleic acid, a single-stranded nucleic acid, or a double-stranded nucleic acid. The nucleic acid may also contain modified nucleotides. The nucleic acid can be used interchangeably with oligonucleotide and polynucleotide.

[0012] As used herein, the term "amplification reaction" broadly refers to a method of replicating and amplifying a specific sequence in a nucleic acid.

[0013] As used herein, the term "ligation" refers to a reaction that forms a phosphodiester bond to connect nucleic acids to each other and can be used synonymously with joining.

[0014] The method of the present embodiment includes a step of performing an amplification reaction on a nucleic acid sample containing a primer containing a partial sequence of gene I and a primer containing a partial sequence of gene II.

[0015] The nucleic acid sample may contain the fusion gene to be detected. If the nucleic acid sample contains a fusion gene, the fusion gene may be included in the nucleic acid sample in any form, for example, as part of DNA, RNA, cell sample, tissue sample, etc., or it may be included in the nucleic acid sample as an amplification product obtained by amplifying the region containing the fusion gene by PCR or the like. If the nucleic acid sample contains a fusion gene, the fusion gene will be detected by the method of this embodiment, and if the nucleic acid sample does not contain a fusion gene, the fusion gene will not be detected by the method of this embodiment. Therefore, the presence or absence of a fusion gene in the nucleic acid sample can be confirmed by the method of this embodiment. When an amplification reaction is carried out on a nucleic acid sample containing primers containing a portion of the sequence of gene I and primers containing a portion of the sequence of gene II, if the nucleic acid sample contains a fusion gene, the fusion gene acts as a template for the amplification reaction, and an amplicon amplified on the template is obtained. In addition to the target nucleic acid, primers containing a portion of the sequence of gene I, and primers containing a portion of the sequence of gene II, the nucleic acid sample may also contain nucleic acids such as dNTPs, buffers, reagents, enzymes such as polymerases, etc.

[0016] When an amplification reaction is carried out in the presence of a primer containing a portion of the sequence of gene I, a primer containing a portion of the sequence of gene II, and a nucleic acid sample, an amplicon containing the sequence of the fusion region of the fusion gene is obtained if the nucleic acid sample contains a fusion gene derived from gene I and gene II. The fusion region of the fusion gene is a region that includes the junction of genes I and II, and may be any region as long as it includes the nucleic acids on both sides of the junction.

[0017] The primer sequence containing a portion of gene I or gene II may be any sequence as long as it contains a portion of gene I or gene II, and its length may also be arbitrary, but the length of the portion containing a portion of gene I or gene II is preferably 10 to 50 nucleotides, and more preferably 15 to 30 nucleotides. Furthermore, the primer containing a portion of gene I or gene II may each consist of a primer consisting of a portion of gene I or gene II. When the purpose is to detect a fusion gene whose chromosome break site is known, the primer sequence can be determined according to the break site, but it is preferable to set the primer to contain a sequence that is included in the fusion gene regardless of the break site. The step of performing an amplification reaction on a nucleic acid sample containing a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II may be a step of amplifying the region containing the sequence of the fusion portion of the fusion gene in the presence of the primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II to obtain an amplicon. Note that if the nucleic acid sample does not contain a fusion gene derived from gene I and gene II, an amplicon containing the sequence of the fusion portion of the fusion gene cannot be obtained. In one embodiment, this embodiment also provides a method for detecting a fusion gene derived from gene I and gene II, which includes the step of amplifying a region containing the sequence of the fusion portion of the fusion gene to obtain an amplicon in the presence of a primer containing a sequence of a portion of gene I and a primer containing a sequence of a portion of gene II.

[0018] Furthermore, it is preferable that the primer containing a portion of the sequence of gene I and the primer containing a portion of the sequence of gene II each further contain a single-stranded nucleic acid region at their 5' end, derived from a sequence obtained by splitting a predetermined sequence into two at an arbitrary position. The predetermined sequence may be any single-stranded sequence and may be of any length, but is preferably 10 to 400 nucleotides, and more preferably 10 to 200 nucleotides. The position where the predetermined sequence is split can be appropriately determined by a person skilled in the art. The single-stranded nucleic acid region contained in each primer, derived from a sequence obtained by splitting a predetermined sequence into two at an arbitrary position, is preferably 5 to 200 nucleotides, and more preferably 5 to 100 nucleotides. Each primer containing a single-stranded nucleic acid region derived from a sequence obtained by splitting a predetermined sequence into two at an arbitrary position is preferably 10 to 200 nucleotides, and more preferably 10 to 100 nucleotides.

[0019] By performing an amplification reaction using the above primers, an amplicon is obtained that contains regions at both ends derived from sequences obtained by splitting a predetermined sequence into two at any position. Here, in this specification, the single-stranded nucleic acid region "derived from" a sequence obtained by splitting a predetermined sequence into two at any position, which is added to the 5' end of each primer, is a region in which the predetermined sequence appears when the amplicon obtained using the primer containing the said region is circularized. For example, if the predetermined sequence is a single-stranded sequence consisting of 5'-AAAATAAAATCCCCGCCCCG-3', and single-stranded nucleic acid regions derived from sequences obtained by splitting the sequence into 10-base pairs are attached to each primer, then 5'-AAAATAAAAT-3' and 5'-CGGGGCGGGG-3', or 5'-CCCCGCCCCG-3' and 5'-ATTTTATTTT-3', are attached to each primer, respectively, so that when the amplicon is circularized, the predetermined sequence appears. Furthermore, for example, if the predetermined sequence is a double-stranded sequence consisting of 5'-AAATCCCG-3' and 5'-CGGGATTT-3', and single-stranded nucleic acid regions derived from sequences obtained by splitting them into 4-base pairs are attached to each primer, then the predetermined sequence will appear when the amplicon is circularized by attaching 5'-AAAT-3' and 5'-CGGG-3', or 5'-CCCG-3' and 5'-ATTT-3', respectively, to each primer.

[0020] The amplification reaction may be carried out by a method known to those skilled in the art, but it is preferable to carry it out by PCR. The PCR method may be multiplex PCR. In this embodiment, when the amplification reaction is carried out by multiplex PCR, it becomes possible to detect two or more fusion genes. The temperature settings, reaction times, reagents used, and other conditions for each stage of the amplification reaction can be appropriately determined by those skilled in the art.

[0021] After the amplification reaction, a process may be performed to degrade unreacted primers and nucleic acids such as dNTPs, other than the amplification reaction product (amplicon). This process may be carried out using enzymes and reagents known to those skilled in the art, but for example, unwanted nucleic acids other than the amplification reaction product can be degraded by cleaving the nucleic acid sequence and dephosphorylating the nucleic acid using exonuclease or alkaline phosphatase. The reaction conditions for each degradation process can be appropriately determined by those skilled in the art. After the amplification reaction, or if a process to degrade unreacted nucleic acids has been performed, a ligation reaction is carried out after the degradation process.

[0022] The method of this embodiment includes the step of performing a ligation reaction on a nucleic acid sample on which an amplification reaction has been performed, in the presence of a crosslinking probe. Here, the nucleic acid sample on which an amplification reaction has been performed refers to the nucleic acid sample containing the amplicon obtained by the amplification reaction, if the nucleic acid sample contains a fusion gene derived from gene I and gene II.

[0023] As used herein, "crosslinking probe" refers to a probe that crosslinks two nucleic acid sequences, preferably a probe that hybridizes to two adjacent nucleic acid sequences. In this embodiment, the crosslinking probe has sequences such that the 5' terminal sequence and the 3' terminal sequence of the amplicon obtained when the nucleic acid sample contains a fusion gene in the amplification reaction step are adjacent to each other and hybridize. That is, the crosslinking probe has adjacent sequences that are complementary to the 5' terminal sequence of the amplicon and complementary to the 3' terminal sequence. The crosslinking probe may be a double-stranded nucleic acid or a single-stranded nucleic acid. The length of the crosslinking probe may be appropriately determined depending on the Tm value of the crosslinking probe and the two nucleic acid sequences that the crosslinking probe hybridizes, the reaction temperature of the ligation reaction, etc., but it is preferably 10 to 400 nucleotides, and more preferably 10 to 200 nucleotides. The sequences of the crosslinking probe that are complementary to the 5' terminal sequence and the 3' terminal sequence of the amplicon are preferably 5 to 200 nucleotides, and more preferably 5 to 100 nucleotides, respectively. In the presence of the crosslinking probe, a ligation reaction is performed on the nucleic acid sample after the amplification reaction has been carried out, thereby joining the 5' and 3' ends of the amplicon hybridized to the crosslinking probe. The amplicon obtained in the above amplification reaction step is an amplicon containing the sequence of the fusion portion of the fusion gene, which is obtained when the nucleic acid sample contains a fusion gene. Therefore, a circular DNA oligo containing the sequence of the fusion portion of the fusion gene is obtained by the ligation reaction. The step of performing a ligation reaction in the presence of the crosslinking probe on the nucleic acid sample after the amplification reaction may also be a step of joining the 5' and 3' ends of the amplicon hybridized to the crosslinking probe in the presence of a ligase to obtain a circular DNA oligo containing the sequence of the fusion portion of the fusion gene. In one embodiment, this embodiment also provides a method for detecting a fusion gene derived from gene I and gene II, which includes the step of joining the 5' and 3' ends of an amplicon hybridized to a crosslinked probe in the presence of a ligase to obtain a circular DNA oligo containing the sequence of the fusion portion of the fusion gene.

[0024] Here, it is preferable to heat the nucleic acid sample that has undergone amplification in the presence of a crosslinking probe before performing the ligation reaction on the sample. If the nucleic acid sample that has undergone amplification contains an amplicon, heating will cause the double-stranded amplicon to separate into a single strand due to thermal denaturation. After heating, rapid cooling may be performed to further denature the double-stranded amplicon. In the step of performing the ligation reaction on the nucleic acid sample that has undergone amplification in the presence of a crosslinking probe, it is preferable that the 5' and 3' ends of the single-stranded amplicon hybridized to the crosslinking probe are joined by ligase. The heating temperature, heating time, and other reaction conditions can be appropriately determined by a person skilled in the art, and any conditions are acceptable as long as the double-stranded amplicon separates into a single strand. In one embodiment, this embodiment also provides a method for detecting fusion genes derived from gene I and gene II, which includes the steps of heating a nucleic acid sample on which an amplification reaction has been carried out, and carrying out a ligation reaction on the heated nucleic acid sample in the presence of a crosslinking probe.

[0025] In the nucleic acid sample of this embodiment, if a fusion gene is present, a circular DNA oligo is obtained by the ligation reaction. Therefore, the presence or absence of the circular DNA oligo can be used to confirm the presence or absence of the fusion gene.

[0026] As used herein, "circular DNA oligo" refers to a DNA oligo in which an amplicon containing the sequence of the fusion portion of a fusion gene is formed into a ring, and is preferably a single-stranded circular DNA oligo. The length of the circular DNA oligo in this embodiment may be arbitrary and varies depending on the length of the amplicon obtained in the amplification reaction.

[0027] In this case, it is preferable that one of the 5' ends of the primer in this embodiment is phosphorylated in order for the 5' and 3' ends of the amplicon to be joined.

[0028] Furthermore, if each primer in the amplification reaction contains a single-stranded nucleic acid region at its 5' end derived from a sequence obtained by splitting a predetermined sequence into two at an arbitrary position, the circular DNA oligo obtained by the ligation reaction contains the predetermined sequence. In this case, it is preferable that the crosslinking probe has a sequence corresponding to part or all of the predetermined sequence, specifically a sequence complementary to part or all of the predetermined sequence. When the above primers are used, the amplicon contains regions at both ends derived from sequences obtained by splitting a predetermined sequence into two at an arbitrary position, and has a sequence such that the predetermined sequence appears when it is circularized. Therefore, because the crosslinking probe has a sequence complementary to part or all of the predetermined sequence, the 5' end sequence and the 3' end sequence of the amplicon hybridize adjacent to the crosslinking probe. The sequence complementary to part or all of the predetermined sequence in the crosslinking probe includes a sequence complementary to the region derived from the predetermined sequence in the 5' end sequence of the amplicon, and a sequence complementary to the region derived from the predetermined sequence in the 3' end sequence. If a crosslinking probe has a sequence that corresponds to part or all of a predetermined sequence, the crosslinking probe may contain any other sequence as long as that sequence is continuous; however, it is preferable that the crosslinking probe consists only of sequences that correspond to part or all of the predetermined sequence.

[0029] Furthermore, it is preferable that the predetermined sequence is not a human-derived sequence. When the crosslinking probe has a sequence that corresponds to part or all of the predetermined sequence that is human-derived, and the target nucleic acid to be detected for the fusion gene is human-derived, the crosslinking probe may hybridize to regions other than the 5' and 3' terminal sequences of the amplicon. Therefore, in order to increase the frequency of hybridization of the 5' and 3' terminal sequences of the amplicon to the crosslinking probe, it is preferable that the predetermined sequence is not a human-derived sequence. When the crosslinking probe has a sequence that corresponds to part or all of the predetermined sequence, and the crosslinking probe also includes other sequences in addition to the predetermined sequence, it is preferable that these other sequences are also not human-derived sequences.

[0030] If the primers in the amplification reaction include a single-stranded nucleic acid region derived from a sequence obtained by splitting a predetermined sequence into two at any position, then a crosslinking probe having a sequence corresponding to part or all of the predetermined sequence can be used in common in the ligation reaction, regardless of the sequence of the region amplified in the amplification reaction. In other words, by having the crosslinking probes in each primer in the amplification reaction and the crosslinking probes in the ligation reaction have sequences derived from or corresponding to the predetermined sequence, it becomes possible to use one type of crosslinking probe for each detected fusion gene.

[0031] In the step of performing a ligation reaction on a nucleic acid sample that has undergone an amplification reaction in the presence of a crosslinking probe, the reaction conditions such as reaction time and processing temperature can be appropriately determined by a person skilled in the art. The ligation reaction may be carried out by hybridizing the crosslinking probe to the amplicon contained in the nucleic acid sample that has undergone an amplification reaction, and then adding ligase to the reaction solution. Alternatively, the hybridization of the crosslinking probe to the amplicon contained in the nucleic acid sample that has undergone an amplification reaction and the ligation reaction may be carried out in parallel in a reaction solution containing the nucleic acid sample that has undergone a ligation reaction, the crosslinking probe, and ligase together. Alternatively, the nucleic acid sample that has undergone an amplification reaction may be added to a reaction solution containing the crosslinking probe and ligase together, and the hybridization of the crosslinking probe to the amplicon contained in the nucleic acid sample that has undergone an amplification reaction and the ligation reaction may be carried out in parallel.

[0032] The method of this embodiment includes a step of analyzing a nucleic acid sample on which a ligation reaction has been performed. In the method of this embodiment, if the nucleic acid sample contains a fusion gene, a circular DNA oligo is obtained, and the presence or absence of the fusion gene can be confirmed by the presence or absence of the circular DNA oligo. The presence or absence of the circular DNA oligo is analyzed by analyzing the nucleic acid sample on which a ligation reaction has been performed. Here, a nucleic acid sample on which a ligation reaction has been performed refers to a nucleic acid sample that contains a circular DNA oligo obtained by the ligation reaction (a circular DNA oligo obtained by joining the 5' and 3' ends of the amplicon obtained by the amplification reaction) if the nucleic acid sample contains a fusion gene derived from gene I and gene II.

[0033] The method for analyzing the presence or absence of circular DNA oligos may be determined as appropriate by those skilled in the art, but analysis by PCR is preferred. When using PCR, the region to be amplified may be determined as appropriate by those skilled in the art, but it is preferable that the region including the junction formed by the ligation reaction is amplified. Since the region including the junction formed by the ligation reaction exists only when a nucleic acid sample contains circular DNA oligos, the presence or absence of circular DNA oligos can be confirmed by amplifying the region including the junction. Therefore, the step of analyzing a nucleic acid sample on which a ligation reaction has been performed may be a step of analyzing the amplification reaction of the region including the ligase junction in the circular DNA oligo. In one embodiment, this embodiment also provides a method for detecting a fusion gene derived from gene I and gene II, which includes a step of analyzing the amplification reaction of a region including a ligase-mediated junction in a circular DNA oligo.

[0034] Analysis of the amplification reaction may be performed by sequencing analysis or electrophoresis analysis of the amplified amplicon, but quantitative real-time PCR analysis is preferred. In the case of quantitative real-time PCR analysis, the detection probe used is preferably a probe that hybridizes to a specific region of the template nucleic acid, and more preferably a probe that is fluorescently labeled and degraded by the 5'→3' exonuclease activity of polymerase to emit fluorescence. When the amplification reaction is analyzed using quantitative real-time PCR, the detection primer sequence used can be arbitrary, as long as it amplifies the region of the circular DNA oligo containing the ligase junction. If each primer used in the amplification reaction contains a single-stranded nucleic acid region at its 5' end derived from a sequence obtained by splitting a predetermined sequence into two at any position, and the circular DNA oligo obtained in the ligation reaction contains the predetermined sequence, then it is preferable that the detection primer used in the quantitative real-time PCR analysis amplifies the region contained in the predetermined sequence. Furthermore, when the detection primer used in quantitative real-time PCR analysis is a detection primer that amplifies a region contained in a predetermined sequence, and the detection probe used is a probe that hybridizes to a specific region of the template nucleic acid, it is preferable to use a detection probe that hybridizes to a region contained in the predetermined sequence of the circular DNA oligo. That is, it is preferable that the detection probe has a sequence complementary to part or all of the predetermined sequence. In this case, the region contained in the predetermined sequence is amplified by polymerase by the detection primer, and the detection probe hybridized to the predetermined sequence is degraded by the 5'→3' exonuclease activity of the polymerase, emitting fluorescence. The presence or absence of the circular DNA oligo can be confirmed by analyzing the emitted fluorescence intensity. Specifically, the presence or absence of the circular DNA oligo can be confirmed from the difference in Ct value (ΔCt) when using a control sample that does not contain the circular DNA oligo.

[0035] By including sequences derived from a predetermined sequence in each primer used in the amplification reaction, it becomes possible to prepare crosslinking probes in the ligation reaction using only sequences corresponding to part or all of the predetermined sequence. Furthermore, it becomes possible to prepare detection primers and detection probes in quantitative real-time PCR analysis to confirm the presence or absence of circular DNA oligos using only sequences corresponding to part or all of the predetermined sequence. By incorporating sequences derived from predetermined sequences into each primer used in the amplification reaction, and by creating crosslinking probes, detection primers, and detection probes using sequences derived from or corresponding to the predetermined sequences, fusion genes can be easily detected without having to prepare a separate detection probe for each fusion gene.

[0036] In one embodiment, this embodiment also provides a method for detecting a fusion gene derived from gene I and gene II, comprising the steps of: amplifying a region containing the sequence of the fusion portion of a fusion gene in the presence of a primer containing a sequence of a portion of gene I and a primer containing a sequence of a portion of gene II to obtain an amplicon; hybridizing the amplicon to a crosslinking probe; junctioning the 5' and 3' ends of the amplicon hybridized to the crosslinking probe in the presence of a ligase to obtain a circular DNA oligo containing the sequence of the fusion portion of a fusion gene; and analyzing the amplification reaction of the region containing the ligase-mediated junction in the circular DNA oligo.

[0037] Since fusion genes can be detected by checking for the presence or absence of circular DNA oligos, this embodiment also provides a method for producing circular DNA oligos.

[0038] The aspects of the method provided as the first embodiment also apply to other embodiments described below.

[0039] In a second embodiment, a method is provided for producing a circular DNA oligo containing the sequence of the fusion portion of a fusion gene derived from gene I and gene II, comprising the steps of: amplifying a region containing the sequence of the fusion portion of the fusion gene to obtain an amplicon in the presence of a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II; hybridizing the amplicon to a crosslinking probe; and joining the 5' and 3' ends of the amplicon hybridized to the crosslinking probe in the presence of a ligase.

[0040] A circular DNA oligo prepared using the method provided in the second embodiment can be used in the method for detecting fusion genes provided in the first embodiment. This embodiment also provides a method for producing circular DNA oligos for detecting fusion genes.

[0041] In a third embodiment, a kit for detecting a fusion gene derived from gene I and gene II is provided, comprising a primer containing a portion of the sequence of gene I, a primer containing a portion of the sequence of gene II, and a crosslinking probe.

[0042] The primers containing a portion of the sequence of gene I, the primers containing a portion of the sequence of gene II, and the crosslinking probe, provided as a kit in this embodiment, may be provided together with any buffer and other necessary reagents. Specifically, they may be provided together with deoxynucleoside triphosphate (dNTP), (NH4)2SO4, Tween-20, MgCl2, water, Tris-HCl, BSA, DMSO, glycerol, DNA or RNA degrading enzyme inhibitors, etc. They may also be provided together with enzymes such as polymerases and ligases necessary for amplification reactions and ligation reactions, and may be provided together with target nucleic acids for the detection of fusion genes.

[0043] The kit of this embodiment may be provided with the primers containing a portion of the sequence of gene I, the primers containing a portion of the sequence of gene II, and the cross-linking probes in the same container, or they may be provided in separate containers. If they are provided in separate containers, those containers may be provided together in one box or the like.

[0044] The kit of this embodiment may be a kit for producing a circular DNA oligo containing the sequence of the fusion portion of a fusion gene derived from gene I and gene II.

[0045] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0046] <Amplification of Multiple Fusion Genes by One-step RT-PCR> As the template nucleic acid, RNA derived from the HEL 299 cell line (also referred to as template RNA) into which COSF571, which is one of the NTRK3 fusion genes, and COSF1326, which is one of the NTRK1 fusion genes, were introduced was used. For the PCR primers for COSF571 and COSF1326, those shown in Table 1-1 were used. A phosphate group modification was introduced at the 5'-end of the forward primer and used for PCR. The primers were designed such that the RT-PCR amplicon amplified by the primers contained the chromosomal breakpoint. Furthermore, as described in the annotation of Table 1-1, the sequences indicated by underlines and double underlines, which were obtained by splitting a certain common sequence, were added to the primers for amplifying each fusion gene. The primer to which the sequence obtained by splitting the common sequence was added is called a common sequence-added primer.

[0047]

Table 1-1

[0048] For the one-step RT-PCR reagent, PrimeScript II High Fidelity One Step RT-PCR Kit (Catalog number R026A) manufactured by TaKaRa Bio was used. For one-step RT-PCR, each primer shown in Table 1-1 was used at a final concentration of 400 nM with respect to the one-step RT-PCR reagent, and 1,000 copies of COSF571 and 100,000 copies of COSF1326 of the template RNA were subjected to one-step RT-PCR. The one-step RT-PCR reaction was carried out using a thermal cycler according to the program shown in Table 1-2. In one-step RT-PCR, by using a common sequence-added primer, sequences shown in Table 1 with underlines and double underlines obtained by splitting the common sequence were added to the 5'-end and 3'-end of an amplicon that is an RT-PCR amplicon, namely, an amplicon of a region containing the chromosomal breakpoint of COSF571 or an amplicon of a region containing the chromosomal breakpoint of COSF1326, respectively.

[0049]

Table 1-2

[0050] <ExoSAP Treatment of RT-PCR Amplicon Solution> After the one-step RT-PCR reaction, 2 μL of ExoSAP-IT Express reagent (hereinafter referred to as ExoSAP reagent) was mixed with 5 μL of the RT-PCR reaction solution and incubated at 37 °C for 4 minutes. The unreacted primers and dNTPs present in the RT-PCR amplicon solution were decomposed and removed by the ExoSAP reagent. Thereafter, it was incubated at 80 °C for 1 minute to inactivate the ExoSAP reagent, and the RT-PCR reaction solution after ExoSAP treatment was diluted 10-fold with water.

[0051] <Circularization by Ligation Reaction> The RT-PCR amplicon and the cross-linked ssDNA oligo were mixed. The RT-PCR amplicon-cross-linked ssDNA oligo mixture was incubated at 95 °C for 5 minutes to dissociate non-specific double-strand formation (non-specific binding of DNAs) by heat denaturation. After rapid cooling on ice, it was incubated at 50 °C for 10 minutes to perform annealing of the RT-PCR amplicon and the cross-linked ssDNA and the joining reaction of the 5'-phosphorylated end and the 3'-end of the RT-PCR amplicon by DNA ligase. The cross-linked ssDNA oligos shown in Table 1-3 were used.

[0052]

Table 1-3

[0053] After the ligation reaction, the enzymatic reaction was stopped with an 80% formamide + 100 mM EDTA solution (at which point the solution was diluted 2.5 times). Subsequently, the ligated reaction solution was diluted 40 times with water to prepare a 100-fold dilution relative to the ligation reaction stage. The RT-PCR amplicon was cyclically formed by the ligation reaction (fully cyclic RT-PCR amplicon).

[0054] <Detection of fusion genes by quantitative real-time PCR> The reaction mixture containing the fully circularized RT-PCR amplicon was mixed with the quantitative real-time PCR master mix and the primer and probe sets shown in Table 1-4 to prepare the qPCR reaction mixture. Real-time PCR was performed using the real-time PCR reaction program shown in Table 1-5.

[0055] [Table 1-4]

[0056] [Table 1-5]

[0057] <Quantitative Real-Time PCR Results> The negative control RT-PCR amplicon, which is not a fully circularized RT-PCR amplicon, is an RT-PCR amplicon that annealed to a gap(+) cross-linked ssDNA oligo during the ligation reaction, resulting in a gap and preventing circularization. Quantitative real-time PCR was performed using a fully circularized RT-PCR amplicon containing a region with a chromosome breakpoint of COSF571, resulting in a ΔCp value of 13.5 compared to the negative control (Figure 1). Similarly, quantitative real-time PCR was performed using a fully circularized RT-PCR amplicon containing a region with a chromosome breakpoint of COSF1326, resulting in a ΔCp value of 10.1 compared to the negative control (Figure 1).

[0058] From the above, it was possible to construct a fully circularized RT-PCR amplicon independent of fusion genes or chromosome breakpoints by adding sequences obtained by splitting a common sequence to the primers used in one-step RT-PCR. This confirmed that fusion genes could be detected simultaneously for multiple parameters.

Claims

1. A method for producing a circular DNA oligo containing the sequence of the fusion portion of a fusion gene derived from gene I and gene II, A step of obtaining an amplicon by amplifying a region containing the sequence of the fusion portion of a fusion gene in the presence of a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II. A process in which an amplicon hybridizes with a crosslinking probe, and A method comprising the step of joining the 5' and 3' ends of an amplicon hybridized to a crosslinking probe in the presence of a ligase.

2. The circular DNA oligo further contains a predetermined sequence, The method according to claim 1, wherein a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II each further include a single-stranded nucleic acid region at their 5' ends derived from a sequence obtained by splitting a predetermined sequence into two at any position.

3. The method according to claim 2, wherein the crosslinking probe has an arrangement corresponding to part or all of a predetermined arrangement.

4. The method according to claim 2, wherein the predetermined sequence is not a human-derived sequence.

5. The method according to claim 2, wherein the length of the predetermined sequence is 10 to 400 nucleotides.

6. The method according to any one of claims 1 to 5, wherein the step of obtaining the amplicon is performed by multiplex PCR.

7. The method according to any one of claims 1 to 5, wherein the fusion gene is a cancer-causing gene.

8. A method for detecting a fusion gene derived from gene I and gene II, A step of performing an amplification reaction on a nucleic acid sample containing a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II. A step of performing a ligation reaction on a nucleic acid sample that has undergone an amplification reaction in the presence of a crosslinking probe, and A method comprising the step of analyzing a nucleic acid sample on which a ligation reaction has been performed.

9. The method according to claim 8, wherein the step of carrying out the amplification reaction is the step of obtaining an amplicon containing the sequence of the fusion portion of the fusion gene.

10. The method according to claim 9, wherein the step of carrying out a ligation reaction in the presence of a crosslinking probe is to join the 5' and 3' ends of an amplicon hybridized to the crosslinking probe in the presence of a ligase to obtain a circular DNA oligo containing the sequence of the fusion portion of the fusion gene.

11. The process of analyzing nucleic acid samples after ligation reactions is The method according to claim 10, comprising the step of analyzing the amplification reaction of a region including a ligase-mediated junction in a circular DNA oligo.

12. The method according to any one of claims 8 to 11, wherein a primer containing a portion of the sequence of gene I and a primer containing a portion of the sequence of gene II each further include a single-stranded nucleic acid region at their 5' ends derived from a sequence obtained by splitting a predetermined sequence into two at any position.

13. The method according to claim 12, wherein the crosslinking probe has an arrangement corresponding to a part or all of a predetermined arrangement.

14. The method according to claim 12, wherein the arrangement of the region including the junction is an arrangement that corresponds to a part or all of a predetermined arrangement.

15. The method according to claim 12, wherein the predetermined sequence is not a human-derived sequence.

16. A kit for detecting fusion genes derived from gene I and gene II, A primer containing a portion of the sequence of gene I, and a primer containing a portion of the sequence of gene II, and A kit including a crosslinking probe.

17. The kit further comprises a primer having a sequence of a portion of a given sequence, A primer containing a portion of the sequence of gene I, and a primer containing a portion of the sequence of gene II, each further include a single-stranded nucleic acid region at its 5' end derived from a sequence obtained by splitting a predetermined sequence into two. The kit according to claim 16, wherein the crosslinking probe has an arrangement corresponding to part or all of a predetermined arrangement.

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