Method for detecting DNA fragment ligation and kit therefor

Abstract

The present disclosure relates to the field of methods and kits for molecular diagnostics and genomics. More specifically, the present disclosure relates to methods and kits for detecting DNA fragment ligation events or distinguishing alternative splicing events. The present disclosure also relates to a method for administering appropriate treatment to a subject by determining the risk of a particular cancer type or genotype.

Description

[Technical Field]

[0001] The present invention relates to the field of methods and kits for molecular diagnostics and genomics. More specifically, the present invention relates to methods and kits for detecting ligation events of DNA fragments or identifying alternative splicing events. The present invention also relates to a method for determining the risk of a specific cancer type or determining the genotype, thereby providing an appropriate treatment to a subject. [Background technology]

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Provisional Application No. 63 / 150,095, filed February 17, 2021, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety into this disclosure. The ASCII copy, created on January 13, 2022, is named "ACTG-9PCT_ST25.txt" and is 16,384 bytes in size.

[0004] In genetics, DNA rearrangements, translocations, tandem repeats, inversions, insertions, deletions, or other chimeric mutations that occur when DNA fragments are joined are often synonymous with disease. This type of DNA damage in the cells of our organs has been proven to be the origin of genetic diseases and cancer. Therefore, detecting DNA fragment joins can be used to screen for potential health disorders or diseases. However, this is still far from being sufficient for precision medicine.

[0005] Constitutive splicing is the removal of introns and the joining of exons during RNA splicing. Alternative splicing is a deviation from normal splicing in which exons are skipped, introns are retained, exons are mutually exclusive, or alternative 5' or 3' splice sites are preserved in the mature mRNA. In recent years, alternative splicing has gained attention for its role in gene expression and its association with disease. For example, numerous intron retention events can be detected in the cytoplasm of primary cancer cells and correlate with the diversity of the cancer cell transcriptome.

[0006] DNA fragment ligation events and alternative splicing events affect the proteins produced and can significantly influence disease risk, disease progression, and drug response. Detecting DNA fragment ligation or distinguishing between these genetic variants and alternative splicing can be used as diagnostic markers and may be important for future targeted therapies in gene-related diseases. The correlation between alternative gene splicing and anticancer drug resistance is discussed in Wang, Bi-Dar, and Norman H. Lee, "Aberrant RNA splicing in cancer and drug resistance," Cancers 10.11 (2018):458, which is incorporated herein by reference.

[0007] Identification of DNA fragment ligation events or specific alternative splicing events can be performed using bioinformatics techniques, such as next-generation sequencing (NGS), immunohistochemistry (IHC), fluorescent in vivo hybridization (FISH), and qRT-PCR, microarray, or RNA-seq data analysis. While NGS provides comprehensive and detailed information, its clinical application is limited due to its cost, time-consuming nature, and the need for larger sample sizes. IHC detects the presence of proteins produced, but it is difficult to distinguish between genotypic variations and phenotypic changes. FISH can detect gene fusions, but it requires separate reactions for each fusion type and highly trained experts to analyze the results. Therefore, novel, more accurate, and comprehensive methods are needed to detect DNA fragment ligation types and predict genotypes, especially for detecting mRNA splicing defects that have been shown to be associated with characteristic disease traits. Summary of the Invention

[0008] The present disclosure provides a method for detecting a DNA fragment ligation event, the method comprising: (a) obtaining DNA in a sample or DNA obtained from extracted RNA; (b) enriching the DNA with a set of oligonucleotides to obtain a target nucleic acid; (c) probing the target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; including; and (d) detecting a signal reflecting binding of the split probe to the target nucleic acid; Includes:

[0009] According to the above, the oligonucleotides of the set are gene-specific primers or gene-specific probes.

[0010] According to the above, in step (b), the DNA is amplified by multiplex PCR using at least two pairs of gene-specific primers.

[0011] According to the above, the method further comprises: (i) confirming the signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) confirming a signal based on the binding of the first split probe to the 5' end of the partner DNA fragment and / or the binding of the second split probe to the 3' end of the target DNA fragment, thereby determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment; or (iii) determining whether the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment through confirming a signal based on the binding of the third split probe to the third DNA fragment and the result of the target nucleic acid from an independent PCR; The method includes determining:

[0012] According to the above, at least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as the upstream DNA fragment.

[0013] According to the above, at least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as the downstream DNA fragment.

[0014] According to the above, certain of the gene-specific primers target DNA fragment junction boundaries.

[0015] According to the above, certain of the gene-specific primers target within a distance of 0 to 80 bp from the DNA fragment junction boundary.

[0016] According to the above, the first split probe or the second split probe targets within a distance of 0 to 40 bp from the DNA fragment junction boundary.

[0017] According to the above, the first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequence thereof.

[0018] According to the above, the second split probe is selected from the group consisting of SEQ ID NOs: 33, 36 and any complementary sequence thereof.

[0019] According to the above, the third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36 and any complementary sequence thereof.

[0020] According to the above, the length of the split probe is 10 to 60 bp.

[0021] According to the above, in step (c) the target nucleic acid is probed with a split probe and a single probe targeting the DNA fragment junction boundary.

[0022] Examples include ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, and A. DNA KAP13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2. ATG7, ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, C.S APZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CH TOP CLCN6 CLIP1 CLIP2 CLTC CNIH4 CNTRL COL25A1 COX5A CPD CREBB P, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, E IF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131 B. FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1 GOLGA4, GON4L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL 1. HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16. KCTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LM NA, LMNTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1 MEF2D, MET, MIR548F1, MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRI P、MRPL24、MSN、MTSS1、MUC2、MYH9、MYO5A、NACC2、NAV1、NBPF20、NCOA4、NFA SC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3. PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM.PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF21 3, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SHKBP1, SIL1, S LC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPECC1L, SPTBN1, SPTBN2, SQSTM1 , SRCIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1.

[0023] According to the above, the target DNA fragment comprises the sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2 and TMPRSS2.

[0024] According to the above, the partner DNA fragment and the target DNA fragment each comprise different sequences from the same gene selected from the group consisting of AR (e.g., ARV7), BCL2L1, BCL2-Like 11 (BIM or BCL2L11), BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2 (CASP-2), CD19, CD44, CXCR3, Cyclin D1 (CCND1), DMP1, CDH1, EGFR (e.g., EGFRvIII), ER (e.g., ESR1 or ESR2), EZH2, FAS, FGFR2, HRAS (H-RAS), IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

[0025] Additionally, the target DNA fragmentation complexes are ACVR2A-AKT3, AFAP1-NTRK1, and AFAP1-NTRK2 、AFAP1-RET、AGAP3-BRAF、AGBL4-NTRK2、AGGF1-RAF1、AKAP13-NTRK3、AKAP 13-RET, AKAP9-BRAF, AKT3-P2RX5, AKT3-PTPRR, AMOTL2-NTRK1, APIP-FGFR2, ARGLU1-NTRK1, ARHGEF11-NTRK1, ARHGEF2-NTRK1, ATG7-RAF1, ATP1B-NT RK1, AXL-MBIP, BAG4-FGFR1, BAIAP2L1-BRAF, BAIAP2L1-MET, BCAN-NTRK1 BCL6-RAF1, BCR-ABL, BCR-FGFR1, BCR-JAK2, BCR-NTRK2, BCR-RET, BRD3-NU TM1, BRD4-NUTM1, BTBD1-NTRK3, CAPZA2-MET, CBR4-ERBB4, CCDC6-BRAF, CCDC6-RET, CCDC6-ROS1, CD74-NRG1, CD74-NRG2, CD74-NTRK1, CD74-ROS1, CD K12-ERBB2, CDK5RAP2-BRAF, CEL-NTRK1, CEP170-AKT3, CHTOP-NTRK1, CLCN6-RAF1, CLIP1-ALK, CLIP1-ROS1, CLIP2-BRAF, CLIP2-MET, CLTC-ALK, CLTC -ROS1, CNTRL-KIT, COL25A1-ALK, COL25A1-FGFR2, COX5A-NTRK3, CPD-ERBB 2, CTRC-NTRK1, CUX1-BRAF, CUX1-FGFR1, CUX1-RET, DCTN1-ALK, DCTN1-MET DLG1-NTRK3, DNAJC8-ERBB2, EIF3E-RSPO2, EML1-NTRK2, EML4-ALK, EML4- BRAF、EML4-NTRK3、EML4-RET、EPHB2-NTRK1、EPS15-BRAF、EPS15-MET、EPS1 5-NTRK1, ERBB2-CDK12, ERBB2-CFB, ERBB2-CNIH4, ERBB2-CTTN, ERBB2-DNAJC7, ERBB2-ENO1, ERBB2-FCGRT, ERBB2-FKBP10, ERBB2-GRB7, ERBB2-GSE1.<h2 style=";text-align:left;direction:ltr">ERBB2-GTF2E2 / SMIM18、ERBB2-IKZF3、ERBB2-KRT20、ERBB2-KRT39、ERBB2-KRTAP1-4、ERBB2-LMNTD1、ERBB2-LTBP4、ERBB2-MAD2L2、ERBB2-MED1、ERBB 2-PARN、ERBB2-PGAP3、ERBB2-POLD4、ERBB2-PPP1R1B、ERBB2-PRDX4、ERBB2-PSMB3、ERBB2-SHKBP1、ERBB2-SLC39A11、ERBB2-SPTBN2、ERBB2-SRCIN1、E RBB2-TADA2A, ERBB2-TATDN1, ERBB2-XBP1, ERBB2-ZAN, ERBB4-AKAP6, ERBB4-FUS, ERBB4-IKZF2, ERBB4-STK11IP, ERC1-BRAF, ERC1-RET, ERC1-ROS1, ESRP1-RAF1, ESR1-CCDC170, ETV6-FGFR3, ETV6-NTRK2, ETV6-NTRK3, ETV6-PDGFRB, ETV6-PRDM16, EZR-ERBB4, EZR-ROS1, FAM131B-BRAF, FAT1-NTRK3, FG FR2-BICC1, FGFR2-TACC3, FGFR3-TACC3, FIP1L1-PDGFRA, FN1-ALK, FN1-ERBB4, FN1-FGFR1, FNDC3B-PIK3CA, FRY-NTRK3, GKAP1-NTRK2, GOLGA4-RAF1, GON4L-NTRK1, GOPC-ROS1, GRHL2-RSPO2, GRIPAP-NTRK1, GTF2-IRD1-ALK, HACL1-RAF1, HIP1-ALK, HNRNPA2B1-NTRK3, IKZF2-ERBB4, IQSEC1-RAF1, IRF2B P2-NTRK1, KANK1-NTRK2, KCTD16-NTRK2, KCTD8-NTRK2, KHDRBS1-NTRK3, KIAA1549-BRAF, KIF5B-ALK, KIF5B-RET, KIF5B-ERBB4, KIT-ANKRD11, KIT-PDGFRA, KIT-SLC4A4, KMT2A-AFF1, KMT2A-CREBBP, KMT2A-DAB2IP, KMT2A-ELL, KMT2A-EPS15, KMT2A-MLLT1, KMT2A-MLLT10, KMT2A-MLLT11, KMT2A-MLLT3KMT2A-MLLT4、KMT2A-SEP6、KMT2A-SEP9、KTN1-ALK、KTN1-RET、LIPI-NTRK1、LMNA-ALK、LMNA-NTRK1、LMNA-RAF1、LRRC71-NTRK1、LRRFIP1-FGFR1、LRRFI P1-MET、LYN-NTRK3、MAGI3-AKT3、MBNL1-RAF1、MEF2D-NTRK1、MET-MET、MIR548F1-NTRK1、MKRN1-BRAF、MPRIP-ALK、MPRIP-NTRK1、MPRIP-RAF1、MPRIP-R ET、MRPL24-NTRK1、MSN-ALK、MSN-ROS1、MTSS1-ERBB2、MUC2-NTRK2、MYH9-ALK、MYO5A-NTRK3、MYO5A-ROS1、NACC2-NTRK2、NAV1-NTRK2、NBPF20-NTRK2、N COA4-RET、NFASC-NTRK1、NOS1AP-NTRK1、NOS1AP-NTRK2、NRG2-CYSTM1、NRG2-UBE2D2、NRIP1-RSPO2、P2RY8-NTRK1、PAIP1-NTRK2、PAN3-NTRK2、PAPD7-R AF1、PDE4DIP-NTRK1、PEAR1-NTRK1、PHF20-NTRK1、PICALM-BRAF、PICALM-RET、PLEKHA6-NTRK1、PML-RARA、PPFIBP1-ALK、PPFIBP1-MET、PPFIBP1-ROS1、 PPL-NTRK1、PRDX1-NTRK1、PRKAR1A-ALK、PRKAR1A-RET、PRKAR1B-ALK、PRKAR1B-BRAF、PRKAR2A-NTRK2、PRPSAP1-NTRK3、PTPRZ1-MET、QKI-NTRK2、QKI-R AF1、RAC1-AKT3、RAF1-ACTR2、RAF1-AGGF1、RAF1-DAZL、RAF1-ESRP1、RAF1-PHC3、RAF1-TMEM40、RAF1-TRAK1、RAF1-ZPR1、RALGPS2-NTRK3、RANBP2-ALK、 RANBP2-FGFR1、RBPMS-NTRK3、RFWD2-NTRK1、RNF213-ALK、RNF213-NTRK1、RRBP1-ALK、RRBP1-RET、SATB1-ALK、SATB1-RET、SCAF11-PDGFRA、SCP2-NTRK1、SCYL3-NTRK1、SDC4-NRG1、SDC4-ROS1、SEC31A-ALK、SHC1-ERBB2、SIL1-NRG2、SLC34A2-MET、SLC34A2-ROS1、SLC45A3-BRAF、SLC45A3-ERG、SLC45A3-FGF R2、SLMAP-NTRK2、SND1-BRAF、SPECC1L-NTRK2、SPECC1L-NTRK3、SPTBN1-ALK、SQSTM1-ALK、SQSTM1-FGFR1、SQSTM1-NTRK1、SQSTM1-NTRK2、SQSTM1-NTRK 3、SRGAP3-RAF1、SRGAP3-SRGAP3-RAF1、SSBP2-NTRK1、STRN-ALK、STRN-NTRK2、STRN-NTRK3、STRN3-BRAF、STRN3-NTRK1、STRN3-NTRK2、STRN3-NTRK3、TBC1D2-NTRK2、TBL1XR1-NRG1、TBL1XR1-PIK3CA、TBL1XR1-RET、TFG-ALK、TFG-MET、TFG-NTRK1、TFG-NTRK3、TFG-RET、TFG-ROS1、TIMP3-ALK、TIMP3-NTRK1 、TKT-ERBB2、TLE4-NTRK2、TMEM106B-BRAF、TMEM106B-ROS1、TMPRSS2-ERG、TMPRSS2-ETV1、TMPRSS2-ETV4、TMPRSS2-ETV5、TNS3-NTRK2、TP53-NTRK1、TP M3-ALK、TPM3-NTRK1、TPM3-ROS1、TPM4-ALK、TPM4-NTRK3、TPR-ALK、TPR-BRAF、TPR-FGFR1、TPR-MET、TPR-NTRK1、TRAF2-NTRK2、TRAK1-RAF1、TRIM24-BR AF、TRIM24-FGFR1、TRIM24-NTRK2、TRIM24-RET、TRIM33-RET、TRIM33-NTRK1、TRIM4-BRAF、TRIM4-MET、TRIM63-NTRK1、UBE2R2-NTRK3、UFD1-NTRK2、USP 13-PIK3CA、VANGL2-NTRK1、VCAN-NTRK2、VCL-ALK、VCL-NTRK2、VIM-NTRK3、VPS18-NTRK3、WHSC1L1-FGFR1、WHSC1L1-NUTM1、WIPF2-ERBB2、WNK2-NTRK2、ZBTB7B-NTRK1 and ZNF710-NTRK3 mutations are selected from the group consisting of.

[0026] According to the above, the third DNA fragment comprises the sequence of a partner gene or a target gene.

[0027] According to the above, in step (b), the DNA is first amplified with gene-specific primers and then amplified with universal primers to obtain the target nucleic acid.

[0028] According to the above, the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, an aminohexyl, and a pyrene.

[0029] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (a) probing a target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is 0 to 80 bp; Contains; (b) detecting a signal reflecting binding of the split probe to the target nucleic acid; (c) (i) confirming a signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment through confirmation of a signal based on the binding of the first split probe to the 5' end of the partner DNA fragment and / or the binding of the second split probe to the 3' end of the target DNA fragment; or (iii) confirming a signal based on the binding of the third split probe to the third DNA fragment, thereby confirming that the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment; determining the and (d) comparing the length of the target nucleic acid with a reference sequence for constitutive splicing for identity; The present invention provides a method for distinguishing between alternative splicing events, including:

[0030] According to the above, the target nucleic acid is amplified using a set of oligonucleotides.

[0031] According to the above, the target nucleic acid is amplified by multiplex PCR using at least two pairs of gene-specific primers.

[0032] According to the above, the method further comprises the step (e) of reconfirming by independent PCR.

[0033] According to the above, at least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as the upstream DNA fragment.

[0034] According to the above, at least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as a downstream DNA fragment.

[0035] According to the above, at least one of said gene-specific primers targets a DNA fragment junction boundary.

[0036] According to the above, the gene-specific primer targets within a distance of 0 to 80 bp from the DNA fragment junction boundary.

[0037] According to the above, the product of the multiplex PCR is then amplified using universal primers to obtain the target nucleic acid.

[0038] According to the above, the distance between the target sites of the first and second split probes and the DNA fragment ligation boundary is 0 to 40 bp.

[0039] According to the above, the length of the split probe is 10 to 60 bp.

[0040] According to the above, in step (a) of the method, the target nucleic acid is probed with a split probe and a single probe targeting a DNA fragment junction boundary.

[0041] According to the above, the partner DNA fragment and the target DNA fragment each contain different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

[0042] According to the above, said alternative splicing event is a BCR-ABL mutation.

[0043] According to the above, the third DNA fragment comprises the sequence of a partner gene or a target gene.

[0044] According to the above, the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, aminohexyl, and pyrene.

[0045] In one aspect, the disclosure provides a method of treating a subject, the method comprising: (a) determining whether a subject is at risk for cancer or a genotype, comprising detecting DNA fragment ligation events in a sample from the subject by the method described in the preceding paragraph, and / or distinguishing alternative splicing events in a sample from the subject by the method described in the preceding paragraph; and (b) administering: (i) a therapeutically effective amount of an siRNA targeting the DNA fragment ligation event and / or the alternative splicing event; (ii) a therapeutically effective amount of an inhibitor of the fusion protein encoded by said DNA fragment ligation event and / or said alternative splicing event; (iii) a therapeutically effective amount of an agent that inhibits the DNA fragment ligation event and / or the fusion protein encoded by the alternative splicing event; (iv) a therapeutically effective amount of an anti-cancer agent selected from the group consisting of cytokines, apoptosis inducers, anti-angiogenic agents, chemotherapeutic agents, radiotherapeutic agents, and anti-cancer immunotoxins; or (v) providing a targeted genome editing treatment in a cell of said subject; Includes:

[0046] According to the above, the DNA fragment ligation event and / or the alternative splicing event may be selected from the group consisting of ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, AKAP13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG7, ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, CAPZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5R AP2, CEL, CEP170, CFB, CHTOP, CLCN6, CLIP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD, CREBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, EIF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1, GOLGA4, GON4L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL1, HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2 , JAK2, KANK1, KCTD16, KCTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LMNTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MA GI3, MBIP, MBNL1, MED1, MEF2D, MET, MIR548F1, MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRIP, MRPL24, MSN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV 1, NBPF20, NCOA4, NFASC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3,PHC3, PHF20, PICALM, PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RE T, RFWD2, RNF213, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SH KBP1, SIL1, SLC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPECC1L, SPTBN1, SPTBN 2, SQSTM1, SRCIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710 and ZPR1.

[0047] According to the above, the ligation event of the DNA fragment appears with the sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2 and TMPRSS2.

[0048] According to the above, the alternative splicing events appear with different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

[0049] According to the above, said DNA fragment joining event or said alternative splicing event is a BCR-ABL mutation.

[0050] According to the above, the alternative splicing event is selected from the group consisting of constitutive splicing, exon skipping, intron retention, mutually exclusive exons, and alternative 5' or 3' splice sites.

[0051] According to the above, the cancer is selected from the group consisting of carcinoma, sarcoma, lymphoma, leukemia, and myeloma.

[0052] According to the above, the cancer is selected from the group consisting of brain cancer, breast cancer, colon cancer, endocrine cancer, esophageal cancer, female reproductive cancer, head and neck cancer, hepatobiliary cancer, kidney cancer, lung cancer, mesenchymal cell neoplasm, prostate cancer, skin cancer, gastric cancer, exocrine pancreatic tumor, and urinary system cancer.

[0053] In other aspects, the present disclosure also provides a kit for detecting a sample having a DNA fragment ligation event and / or an alternative splicing event, the kit comprising: (a) a set of oligonucleotides; (b) a split probe having: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target sites of the split probes on the target nucleic acid is 0 to 80 bp; and (c) a probe hybridization assay for detecting hybridization signals of split probes, wherein the probe hybridization assay comprises a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, an aminohexyl, and a pyrene; Includes:

[0054] According to the above, the oligonucleotides of the set are gene-specific primers or gene-specific probes.

[0055] According to the above, the kit comprises at least two pairs of gene-specific primers.

[0056] According to the above, the gene-specific primer is designed to obtain the target nucleic acid from the partner DNA fragment as an upstream DNA fragment.

[0057] According to the above, the gene-specific primer is designed to obtain the target nucleic acid from the partner DNA fragment as a downstream DNA fragment.

[0058] According to the above, the kit further comprises a universal primer.

[0059] According to the above, at least one of said gene-specific primers targets a DNA fragment junction boundary.

[0060] According to the above, the gene-specific primer targets within a distance of 0 to 80 bp from the DNA fragment junction boundary.

[0061] According to the above, the first split probe or the second split probe targets within a distance of 0 to 40 bp from the DNA fragment ligation boundary.

[0062] According to the above, the first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequence thereof.

[0063] The second split probe is selected from the group consisting of SEQ ID NOs: 33, 36, and any complementary sequence thereof.

[0064] According to the above, the third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequence thereof.

[0065] In certain other aspects, the present disclosure provides kits comprising the split probes having a length of 10 to 60 bp.

[0066] According to the above, the kit further comprises a single probe targeting a DNA fragment junction boundary.

[0067] ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, and AKA P13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG 7. ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, CAPZ A2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CHTOP. CLCN6, CLIP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD, CREBBP, CT RC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, EIF3E. ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131B, FAT 1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1, GOLG A4, GON4L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL1, HIP 1. HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16, KCTD8. KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LMN TD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1, MEF2D MET, MIR548F1, MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRIP, MRPL 24. MSN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV1, NBPF20, NCOA4, NFASC, NOS1 AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PA RN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM, PLEKHA6.PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF213, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SHKBP1, SIL1, SLC34A2 , SLC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPECC1L, SPTBN1, SPTBN2, SQSTM1, SRCI It is complementary to the sequence of a partner gene selected from the group consisting of N1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1.

[0068] According to the above, the second split probe is complementary to the sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2 and TMPRSS2.

[0069] According to the above, the first split probe and the second split probe are complementary to the partner DNA fragment and the target DNA fragment, respectively, which contain different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

[0070] According to the above, said DNA fragment joining event or said alternative splicing event is a BCR-ABL mutation.

[0071] According to the above, the third split probe is complementary to the third DNA fragment having the sequence of the partner gene or the target gene. [Brief explanation of the drawings]

[0072] The present disclosure will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings, in which:

[0073] [Figure 1] Figure 1 is a schematic diagram of a method for detecting MET gene mutations according to one embodiment of the present invention, which is based on a target-probe hybridization assay in a one-step PCR.

[0074] [Figure 2] 2 is a schematic diagram of a method for detecting NTRK gene mutations according to one embodiment of the present invention, which is based on a two-step PCR target-probe hybridization assay.

[0075] [Figure 3]3 is a schematic diagram of a method for detecting EGFR gene mutations according to one embodiment of the present invention, which is based on a two-step PCR target-probe hybridization assay.

[0076] [Figure 4] Figure 4 shows the array of probe spots printed on the wells of the plate. The numbers in the first row from the top and first column from the left are shown as reference coordinates. The squares labeled 1-117 represent split probe spots, the squares labeled IC001-IC009 represent control probe spots, and the square labeled R144 represents an anchor probe spot.

[0077] [Figure 5] FIG. 5 shows an ETV6-NTRK3 (exon 5 and exon 14) fusion-positive array of probe spots in the wells of a plate according to one embodiment of the present invention.

[0078] [Figure 6] FIG. 6 shows a QKI-NTRK2 (exon 6 and exon 16) fusion-positive array of probe spots in the wells of a plate according to one embodiment of the present invention.

[0079] [Figure 7] FIG. 7 shows an AFAP1-NTRK1 (exon 4 and exon 10) fusion-positive array of probe spots in the wells of a plate according to one embodiment of the present invention.

[0080] [Figure 8] FIG. 8 shows an artificial novel PPL-NTRK3 (exon 22 and exon 14) fusion positive array of probe spots in the wells of a plate according to one embodiment of the present invention.

[0081] [Figure 9]FIG. 9 shows fusion-negative and fusion-positive arrays of probe spots in wells of the control group (water, fusion-negative sample) and the fusion group (PPL-NTRK3). DETAILED DESCRIPTION OF THE INVENTION

[0082] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0083] definition

[0084] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0085] The term "DNA fragment ligation" refers to DNA rearrangements, translocations, tandem repeats, inversions, insertions, deletions, or other chimeric mutations caused by the cutting and religation of DNA fragments. DNA fragment ligation occurs when a hybrid DNA fragment is created from two or more normally separate DNA fragments. The term "DNA fragment ligation" also refers to the synthesis of a cDNA product from mRNA that has followed alternative splicing pathways.

[0086] The term "alternative splicing" refers to the event where a primary transcript can be spliced into two or more isoforms of mRNA. Various categories of alternative splicing have been described, including constitutive splicing, exon skipping, intron retention, mutually exclusive exons, alternative 5' or 3' splice sites, etc.

[0087] The term "target DNA fragment" refers to any nucleic acid molecule, polynucleotide sequence, or any fragment containing a portion of a specific gene or locus in genomic DNA. The term "partner DNA fragment" refers to a fragment whose 3' or 5' sequence is linked to the 5' or 3' sequence of a "target DNA fragment." The target DNA fragment or the partner DNA fragment may include an intact gene, an exon or intron, a regulatory sequence, or any intergenic region. The DNA fragment at the 5' end of the hybrid DNA fragment is referred to as the "upstream DNA fragment," and the DNA fragment at the 3' end of the hybrid DNA fragment is referred to as the "downstream DNA fragment."

[0088] Hybrid DNA fragments have a "DNA fragment ligation boundary," which is the region where one DNA fragment is ligated to another DNA fragment. For example, the region where a partner DNA fragment is ligated to a target DNA fragment, or the region where a partner DNA fragment is ligated to another DNA fragment or fusion junction. The ligation between two or more specific DNA fragments is further multiplied by the DNA fragment ligation boundary.

[0089] Sometimes, a partner DNA fragment consisting of a specific gene sequence and a target DNA fragment may combine in an abnormal combination, resulting in a gene fusion. The term "gene fusion" refers to a phenomenon in which a first gene on a chromosome fuses with a second gene on the same chromosome or a different chromosome, resulting in a hybrid or fusion gene. This phenomenon is also commonly referred to as "gene translocation" or "gene rearrangement." For example, when an NTRK gene is one of multiple fused genes, such a gene fusion is called an "NTRK gene fusion" or "NTRK fusion."

[0090] The gene at the 5' end of a fusion gene is called the "5' gene," and the gene at the 3' end of a fusion gene is called the "3' gene." Fusion genes have a "fusion junction," the site where the genes are fused. The fusion junction is located in the fusion region, defined by a fusion sequence (also called a fusion junction sequence) that includes sequences from the 5' gene and the 3' gene. Different fusion gene combinations result in different "fusion types." Because the fusion junction can be located anywhere within the fusion gene, the fusion between two specific genes is further diversified by the fusion junction. For example, a fusion of the first exon of a first gene with the second exon of a second gene is one fusion type, and a fusion of the third exon of a first gene with the first exon of a second gene is another fusion type.

[0091] Gene fusions may be detected by identifying fusion junctions in DNA or RNA transcripts of that DNA. In the present disclosure, a "fusion type" refers to a unique fusion present in an RNA transcript. In other words, fusions between two specific genes that occur at different sites within the same intron region are considered to be the same fusion type. For example, a fusion between exon 3 of gene A and exon 5 of gene B may have a DNA fusion region that includes a small portion of the intron between exons 3 and 4 of gene A and a large portion of the intron between exons 4 and 5 of gene B. Alternatively, such a fusion may have a DNA fusion region that includes a large portion of the intron between exons 3 and 4 of gene A and a small portion of the intron between exons 4 and 5 of gene B. Although these two fusions have different DNA fusion junctions, they are considered to be the same "fusion type" because the RNA transcripts produced from the two fusions are identical.

[0092] The term "oligonucleotide set" refers to a set of synthetic single-stranded oligonucleotides that can be used to enrich a target gene region for sequencing. In the present disclosure, the terms "gene-specific primer pair," "MET mutation-specific primer pair," "NTRK fusion-specific primer pair," or "EGFRvIII mutation-specific primer pair" refer to DNA primers designed to amplify a target DNA containing a DNA fragment junction boundary or fusion junction. In the present disclosure, the term "gene-specific probe" refers to a synthesized oligonucleotide probe (as bait) complementary to the target gene sequence.

[0093] In the present disclosure, the term "universal primer" refers to a DNA primer designed to amplify any DNA containing the nucleotide sequence of the universal primer. The universal primers are used in pairs, including a universal forward primer and a universal reverse primer.

[0094] Unless otherwise specified, the term "split probe" refers to two or more synthetic single-stranded DNA oligonucleotides capable of hybridizing to DNA fragment junction regions derived from partner DNA fragments and target DNA fragments and / or other DNA fragments.

[0095] Each of the genes described in this disclosure corresponds to a "gene name (or symbol)" listed in the NCBI Gene Database (https: / / www.ncbi.nlm.nih.gov / gene / ). The NCBI Gene Database is therefore used to identify the sequence of the gene or synonyms of the gene name.

[0096] In the present disclosure, a method for detecting a DNA fragment ligation event is provided, the method comprising: (a) obtaining DNA in a sample or DNA obtained from extracted RNA; (b) enriching the DNA with a set of oligonucleotides to obtain amplified target nucleic acids; (c) probing the amplified target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to another DNA fragment, wherein the gaps between the target sites of the split probes on the amplified target nucleic acid are within a distance of 0 to 80 bp from each other; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to another DNA fragment, wherein the gap between the target sites of the split probes on the target nucleic acid is within a distance of 0 to 80 bp; including; and (d) detecting a signal reflecting binding of the split probe to the target nucleic acid; Includes:

[0097] In some embodiments, RNA is prepared from a biological sample. The biological sample can be any sample obtained from an animal or human subject. Examples of biological samples include formalin-fixed, paraffin-embedded (FFPE) tissue sections, peripheral blood mononuclear cells (PBMCs), blood, plasma, or other cells or bodily fluids. In some embodiments, the biological sample is derived from a cancer patient. In some embodiments, the biological sample is derived from a carcinoma, sarcoma, lymphoma, leukemia, or myeloma. In some embodiments, the biological sample is derived from a brain cancer, breast cancer, colon cancer, endocrine cancer, esophageal cancer, female reproductive cancer, head and neck cancer, hepatobiliary cancer, kidney cancer, lung cancer, mesenchymal cell neoplasm, prostate cancer, skin cancer, gastric cancer, exocrine pancreatic tumors, and urinary system cancer patient.

[0098] Preparation of total RNA from biological samples can be performed by various methods known in the art. One typical procedure involves RNA extraction with an organic solvent such as phenol / chloroform and precipitation by centrifugation. Commercially available kits are also available for RNA isolation or purification. Once RNA is obtained, a step called reverse transcription is performed, in which cDNA is produced from the template RNA using reverse transcriptase in conjunction with the four deoxyribonucleoside triphosphates (dNTPs, including dATP, dCTP, dTTP, and dGTP). Reverse transcription may be performed using the SuperScript cDNA synthesis kit (Cat No: 11754050, Invitrogen).

[0099] In some embodiments, the methods of the present disclosure include: (i) confirming a signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment through confirmation of a signal based on binding of the first split probe to the 5' end of the partner DNA fragment and / or binding of the second split probe to the 3' end of the target DNA fragment; or (iii) whether the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment through confirming the signal based on the binding of the third split probe to the third DNA fragment and the result of the target nucleic acid from an independent PCR; The method further includes determining:

[0100] Also provided in the present disclosure is a method for distinguishing between alternative splicing events, the method comprising: (a) probing a target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to another DNA fragment, wherein the gaps between the target sites of the split probes on the target nucleic acid are within a distance of 0 to 80 bp from each other; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to another DNA fragment, wherein the gaps between the target sites of the split probes on the target nucleic acid are within a distance of 0 to 80 bp from each other; having; (b) detecting a signal reflecting binding of the split probe to the target nucleic acid; (c) (i) confirming a signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment through confirmation of a signal based on binding of the first split probe to the 5' end of the partner DNA fragment and / or binding of the second split probe to the 3' end of the target DNA fragment; or (iii) confirming a signal based on the binding of the third split probe to the third DNA fragment, thereby confirming that the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment; determining the and (d) comparing the length of the target nucleic acid with a reference sequence for constitutive splicing for identity; Includes:

[0101] In some embodiments, the oligonucleotides of the set are gene-specific primers or gene-specific probes.

[0102] In some embodiments, the target nucleic acid to be amplified is amplified by multiplex PCR using at least two pairs of gene-specific primers.

[0103] In some embodiments, the method described in the preceding paragraph further comprises reconfirming by independent PCR (eg, Sanger PCR or qPCR).

[0104] In some embodiments, the DNA is amplified using a DNA polymerase and at least two pairs of gene-specific primers to obtain amplified target nucleic acids for probe detection. In some embodiments, the gene-specific primers are NTRK fusion-specific primers, MET mutation-specific primers, or EGFRvIII mutation-specific primers. This amplification may be performed using a multiplex PCR kit (Cat No: 206143, Qiagen) containing DNA polymerase. The gene-specific primers may be provided as reagents before use. In some embodiments, one NTRK fusion-specific primer is used to amplify each target nucleic acid. In some embodiments, two or more pairs of NTRK fusion-specific primers are pooled together to amplify each target nucleic acid.

[0105] In some embodiments, two or more pairs of gene-specific primers consisting of the NTRK fusion-specific primer, the MET mutation-specific primer, and the EGFRvIII mutation-specific primer are pooled together to amplify each target nucleic acid.

[0106] In some other embodiments, gene-specific primers are partially pooled to form a plurality of pooled reagents, each of which contains at least one pair of gene-specific primers.Therefore, the number of pooled reagents can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.In some preferred embodiments, more than 100 pairs of NTRK fusion-specific primers are provided in the form of four pooled reagents, so as to be used in four multiplex amplification reactions.It has been demonstrated that DNA amplification in this manner shows significantly higher performance than a single multiplex amplification reaction that uses all gene-specific primers, and this is probably due to the reduced complexity of primers.

[0107] In some preferred embodiments, the DNA is first amplified using two or more pairs of NTRK fusion-specific primers, and then amplified using a universal primer to obtain an amplified target nucleic acid. When universal primers are used in the disclosed methods, the gene-specific forward primer in each pair of gene-specific primers further comprises the nucleotide sequence of the universal forward primer in the universal primer pair, and the gene-specific reverse primer in each pair of gene-specific primers further comprises the nucleotide sequence of the universal reverse primer in the universal primer pair. The use of universal primers can increase the final yield of possible amplification products, regardless of which DNA fragment is to be detected or which gene-specific primers are used in the first round of amplification. An additional advantage of using universal primers is that when modifying primers to make them detectable, only two, or even only one, universal primer needs to be modified. For example, this can be achieved by linking one universal primer to a connector (e.g., biotin) so that a detectable molecule can be linked to the universal primer. Without using universal primers, all gene-specific primers must be modified, which makes the primer modification step more complex and costly.

[0108] In some preferred embodiments, the amplified target nucleic acid is mixed with a split probe to allow the formation of a probe binding product that reflects binding between the split probe and the amplified target nucleic acid through nucleic acid hybridization.

[0109] In some embodiments, the split probes are specifically designed to direct against a particular sequence of the partner DNA fragment, the target DNA fragment, or the third DNA fragment, so that the exact DNA fragment ligation event can be determined by detecting the signal of the first split probe, the second split probe, or the third split probe from the specific probe ligation product.

[0110] In some embodiments, the length of each split probe is 10 to 60 bp, and in some embodiments, the length of the target nucleic acid is 200 bp or less.

[0111] In some preferred embodiments, the DNA is amplified using at least two pairs of gene-specific primers designed to obtain the target nucleic acid from a partner DNA fragment as the upstream DNA fragment.

[0112] In some preferred embodiments, the DNA is amplified using at least two pairs of gene-specific primers designed to obtain the target nucleic acid from a partner DNA fragment as the downstream DNA fragment.

[0113] In some embodiments, detectable DNA fragment linkages include, but are not limited to, rearrangements, translocations, tandem repeats, inversions, insertions, deletions, or other chimeric mutational events in a sample.

[0114] In some embodiments, genetic mutations that can be detected include those of the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2. and a target gene selected from ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, AKAP13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG7, ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, CAPZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CHTOP, CLCN6, CL IP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD, CREBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, EIF3E, ELL, EML 1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1, GOLGA4, GON4 L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2IRD1, HACL1, HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16, KCTD8, KHDRBS1, KIAA15 49, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LMNTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1, MEF2D, MET, MIR548F1,MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRIP, MRPL24, MSN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV1, NBPF20, NCOA4, NFASC , NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, P HC3, PHF20, PICALM, PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF213, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, S EC31A, SEP6, SEP9, SHC1, SHKBP1, SIL1, SLC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SND1, SPECC1L, SPTBN1, SPTBN2, SQSTM1, S RCIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40 , TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1.

[0115] These include the DNA fragment. ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, A GGF1, AKAP13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, AR HGEF2, ATG7, ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, B TBD1, CAPZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170. CFB, CHTOP, CLCN6, CLIP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD CREBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DN AJC8, EIF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR. FAM131B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS 、GKAP1、GOLGA4、GON4L、GOPC、GRB7、GRHL2、GRIPAP、GSE1、GTF2IRD1、HACL1 HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16, K CTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMN A. LMNTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1 MEF2D, MET, MIR548F1, MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRI P、MRPL24、MSN、MTSS1、MUC2、MYH9、MYO5A、NACC2、NAV1、NBPF20、NCOA4、NFA SC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3. PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM.PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF 213, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SHKBP1, SIL 1, SLC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SND1, SPECC1L, SPTBN1, SPTBN2, SQSTM1, SR The sequence of a partner gene is selected from the group consisting of CIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1.

[0116] In some embodiments, the target DNA fragment comprises the sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2.

[0117] In some embodiments, the third DNA fragment comprises a sequence of a partner gene or a target gene.

[0118] In some embodiments, detectable genetic variants include ACVR2A-AKT3, AFAP1-NTRK1, AFAP1-NTRK2, AFAP1-RET, AGAP3-BRAF, AGBL4-NTRK2, AGGF1-RAF1, AKAP13-NTRK3, AKAP13-RET, AKAP9-BRAF, AKT3-P2RX5, AKT3-PTPRR, AMOTL2-NTRK1, APIP-FGFR2, ARGLU1-NTRK1, ARHGEF11-NTRK1, ARHGEF2-NTRK1, ATG7-RAF1, ATP1B -NTRK1, AXL-MBIP, BAG4-FGFR1, BAIAP2L1-BRAF, BAIAP2L1-MET, BCAN-NTRK1, BCL6-RAF1, BCR-ABL, BCR-FGFR1, BCR-JAK2, BCR-NTRK2, BCR-RET, BRD3 -NUTM1, BRD4-NUTM1, BTBD1-NTRK3, CAPZA2-MET, CBR4-ERBB4, CCDC6-BRAF, CCDC6-RET, CCDC6-ROS1, CD74-NRG1, CD74-NRG2, CD74-NTRK1, CD74-ROS1, CDK12-ERBB2, CDK5RAP2-BRAF, CEL-NTRK1, CEP170-AKT3, CHTOP-NTRK1, CLCN6-RAF1, CLIP1-ALK, CLIP1-ROS1, CLIP2-BRAF, CLIP2-MET, CLTC-ALK, CL TC-ROS1, CNTRL-KIT, COL25A1-ALK, COL25A1-FGFR2, COX5A-NTRK3, CPD-ERBB2, CTRC-NTRK1, CUX1-BRAF, CUX1-FGFR1, CUX1-RET, DCTN1-ALK, DCTN1-ME T, DLG1-NTRK3, DNAJC8-ERBB2, EIF3E-RSPO2, EML1-NTRK2, EML4-ALK, EML4-BRAF, EML4-NTRK3, EML4-RET, EPHB2-NTRK1, EPS15-BRAF, EPS15-MET, EPS 15-NTRK1, ERBB2-CDK12, ERBB2-CFB, ERBB2-CNIH4, ERBB2-CTTN, ERBB2-DNAJC7, ERBB2-ENO1, ERBB2-FCGRT, ERBB2-FKBP10, ERBB2-GRB7, ERBB2-GSE1,ERBB2-IKZF3、ERBB2-KRT20、ERBB2-KRT39、ERBB2-KRTAP1-4、ERBB2-LMNTD1、ERBB2-LTBP4、ERBB2-MAD2L2、ERBB2-MED1、ERBB2-PARN、ERBB2-PGAP3E RBB2-POLD4, ERBB2-PPP1R1B, ERBB2-PRDX4, ERBB2-PSMB3, ERBB2-SHKBP1, ERBB2-SLC39A11, ERBB2-SPTBN2, ERBB2-SRCIN1, ERBB2-TADA2A, ERBB2-TAT DN1, ERBB2-XBP1, ERBB2-ZAN, ERBB4-AKAP6, ERBB4-FUS, ERBB4-IKZF2, ERBB4-STK11IP, ERC1-BRAF, ERC1-RET, ERC1-ROS1, ESRP1-RAF1, ESR1-CCDC170 ETV6-FGFR3 ETV6-NTRK2 ETV6-NTRK3 ETV6-PDGFRB ETV6-PRDM16 EZR- ERBB4, EZR-ROS1, FAM131B-BRAF, FAT1-NTRK3, FGFR2-BICC1, FGFR2-TACC3. FGFR3-TACC3, FIP1L1-PDGFRA, FN1-ALK, FN1-ERBB4, FN1-FGFR1, FNDC3B-P IK3CA, FRY-NTRK3, GKAP1-NTRK2, GOLGA4-RAF1, GON4L-NTRK1, GOPC-ROS1. GRHL2-RSPO2, GRIPAP-NTRK1, GTF2IRD1-ALK, HACL1-RAF1, HIP1-ALK, HNRNPA2B1-NTRK3, IKZF2-ERBB4, IQSEC1-RAF1, IRF2BP2-NTRK1, KANK1-NTRK2K CTD16-NTRK2, KCTD8-NTRK2, KHDRBS1-NTRK3, KIAA1549-BRAF, KIF5B-ALK, KIF5B-RET, KIF5B-ERBB4, KIT-ANKRD11, KIT-PDGFRA, KIT-SLC4A4, KMT2A-A FF1, KMT2A-CREBBP, KMT2A-DAB2IP, KMT2A-ELL, KMT2A-EPS15, KMT2A-MLLT 1, KMT2A-MLLT10, KMT2A-MLLT11, KMT2A-MLLT3, KMT2A-MLLT4, KMT2A-SEP6KMT2A-SEP9、KTN1-ALK、KTN1-RET、LIPI-NTRK1、LMNA-ALK、LMNA-NTRK1、LMNA-RAF1、LRRC71-NTRK1、LRRFIP1-FGFR1、LRRFIP1-MET、LYN-NTRK3、MAGI3- AKT3、MBNL1-RAF1、MEF2D-NTRK1、MET-MET、MIR548F1-NTRK1、MKRN1-BRAF、MPRIP-ALK、MPRIP-NTRK1、MPRIP-RAF1、MPRIP-RET、MRPL24-NTRK1、MSN-ALK 、MSN-ROS1、MTSS1-ERBB2、MUC2-NTRK2、MYH9-ALK、MYO5A-NTRK3、MYO5A-ROS1、NACC2-NTRK2、NAV1-NTRK2、NBPF20-NTRK2、NCOA4-RET、NFASC-NTRK1、NO S1AP-NTRK1、NOS1AP-NTRK2、NRG2-CYSTM1、NRG2-UBE2D2、NRIP1-RSPO2、P2RY8-NTRK1、PAIP1-NTRK2、PAN3-NTRK2、PAPD7-RAF1、PDE4DIP-NTRK1、PEAR1 -NTRK1、PHF20-NTRK1、PICALM-BRAF、PICALM-RET、PLEKHA6-NTRK1、PML-RARA、PPFIBP1-ALK、PPFIBP1-MET、PPFIBP1-ROS1、PPL-NTRK1、PRDX1-NTRK1、P RKAR1A-ALK、PRKAR1A-RET、PRKAR1B-ALK、PRKAR1B-BRAF、PRKAR2A-NTRK2、PRPSAP1-NTRK3、PTPRZ1-MET、QKI-NTRK2、QKI-RAF1、RAC1-AKT3、RAF1-ACTR 2、RAF1-AGGF1、RAF1-DAZL、RAF1-ESRP1、RAF1-PHC3、RAF1-TMEM40、RAF1-TRAK1、RAF1-ZPR1、RALGPS2-NTRK3、RANBP2-ALK、RANBP2-FGFR1、RBPMS-NTRK 3、RFWD2-NTRK1、RNF213-ALK、RNF213-NTRK1、RRBP1-ALK、RRBP1-RET、SATB1-ALK、SATB1-RET、SCAF11-PDGFRA、SCP2-NTRK1、SCYL3-NTRK1、SDC4-NRG1、SDC4-ROS1、SEC31A-ALK、SHC1-ERBB2、SIL1-NRG2、SLC34A2-MET、SLC34A2-ROS1、SLC45A3-BRAF、SLC45A3-ERG、SLC45A3-FGFR2、SLMAP-NTRK2、SND1 -BRAF、SPECC1L-NTRK2、SPECC1L-NTRK3、SPTBN1-ALK、SQSTM1-ALK、SQSTM1-FGFR1、SQSTM1-NTRK1、SQSTM1-NTRK2、SQSTM1-NTRK3、SRGAP3-RAF1、SR GAP3-SRGAP3-RAF1, SSBP2-NTRK1, STRN-ALK, STRN-NTRK2, STRN-NTRK3, STRN3-BRAF, STRN3-NTRK1, STRN3-NTRK2, STRN3-NTRK3, TBC1D2-NTRK2, TBL1XR1-NRG1, TBL1XR1-PIK3CA, TBL1XR1-RET, TFG-ALK, TFG-MET, TFG-NTRK1, TFG-NTRK3, TFG-RET, TFG-ROS1, TIMP3-ALK, TIMP3-NTRK1, TKT-ERBB2, TLE4-NTRK2、TMEM106B-BRAF、TMEM106B-ROS1、TMPRSS2-ERG、TMPRSS2-ETV1、TMPRSS2-ETV4、TMPRSS2-ETV5、TNS3-NTRK2、TP53-NTRK1、TPM3-ALK、T PM3-NTRK1、TPM3-ROS1、TPM4-ALK、TPM4-NTRK3、TPR-ALK、TPR-BRAF、TPR-FGFR1、TPR-MET、TPR-NTRK1、TRAF2-NTRK2、TRAK1-RAF1、TRIM24-BRAF、TR IM24-FGFR1、TRIM24-NTRK2、TRIM24-RET、TRIM33-RET、TRIM33-NTRK1、TRIM4-BRAF、TRIM4-MET、TRIM63-NTRK1、UBE2R2-NTRK3、UFD1-NTRK2、USP13 -PIK3CA、VANGL2-NTRK1、VCAN-NTRK2、VCL-ALK、VCL-NTRK2、VIM-NTRK3、VPS18-NTRK3、WHSC1L1-FGFR1、WHSC1L1-NUTM1、WIPF2-ERBB2、WNK2-NTRK2、Examples include, but are not limited to, ZBTB7B-NTRK1 or ZNF710-NTRK3.

[0119] In one preferred embodiment, detectable NTRK gene fusions include TFG-NTRK1, ETV6-NTRK3, QKI-NTRK2, TPM3-NTRK1, ETV6-NTRK2, TFG-NTRK3, and NACC2-NTRK2. In another preferred embodiment, detectable NTRK gene fusions include PDE4DIP-NTRK1, TRIM63-NTRK1, GON4L-NTRK1, and CTRC-NTRK1.

[0120] In one preferred embodiment, the detectable DNA fragment ligation event includes a target DNA fragment containing an NTRK gene being the downstream DNA fragment and a partner DNA fragment containing a partner gene described in the preceding paragraph being the upstream DNA fragment.

[0121] In one preferred embodiment, the detectable DNA fragment ligation event includes a target DNA fragment containing the EGFR gene being the upstream DNA fragment and a partner DNA fragment containing the partner gene described in the preceding paragraph being the downstream DNA fragment.

[0122] In some embodiments, detectable genetic mutations of alternative splicing events include AR (e.g., ARV7), BCL2L1, BCL2-Like 11 (BIM or BCL2L11), BCOR, BCR-ABL, BIN1, BRAF, BRCA1, BRCA2, CASP2 (CASP-2), CD19, CD44, CXCR3, Cyclin D1 (CCND1), DMP1, CDH1 (E-cadherin), EGFR (e.g., EGFRvIII), ER (e.g., ESR1 or ESR2), EZH2, FAS, FGFR2, HRAS (H-RAS), IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB (e.g., RPS6KB1 or RPS6KB2), STAT3, TP53, TSC2, or VEGF.

[0123] In some embodiments, the first split probe is complementary to a partner DNA fragment having the sequence of a partner gene described in the preceding paragraph and any complementary sequences thereof. In certain other embodiments, the second split probe is complementary to a target DNA fragment having the sequence of a target gene described in the preceding paragraph and any complementary sequences thereof. In certain other embodiments, the third split probe is complementary to a third DNA fragment having the sequence of a partner gene or target gene described in the preceding paragraph and any complementary sequences thereof.

[0124] Table 1 lists split probes specifically designed to detect specific DNA fragment ligation. Each of these probes is designed to be specific to a target region of the DNA fragment (e.g., a target gene or a partner gene) and can be used universally for various DNA fragment types. This allows for an easy probe modification process and allows for accurate determination of DNA fragment ligation events.

[0125] [Table 1]

[0126] In some embodiments, for the detection of NTRK gene fusions, a single probe having either the sequence 5'-GGGAGAATAGCAGGTCCCGT-3' (SEQ ID NO: 31) or 5'-TGGTGTATTAGGCCCAGCCT-3' (SEQ ID NO: 34) is used to compare its detection sensitivity and specificity with split probes having either the sequences of SEQ ID NOs: 32, 33, 35, and 36 (see Table 2, Figure 5, and Figure 6).

[0127] [Table 2]

[0128] In some embodiments, a single target nucleic acid to be amplified is amplified and probed, followed by detection of a single DNA fragment ligation event. In certain other embodiments, two or more target nucleic acids with different sequences can be amplified in a single reaction (called a multiplex amplification reaction) and / or probed in a single reaction (called a multiplex hybridization reaction), allowing simultaneous detection of multiple NTRK fusions. In some embodiments, at least two sets of split probes are selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequences thereof. The probes may be provided as a pooled single reagent or as separate reagents.

[0129] Typically, probes and amplification products are mixed at a specific temperature to promote probe hybridization. The optimal temperature and mixing conditions for probe hybridization vary depending on the probe sequence. Therefore, for multiplex reactions in which at least two probes are used, it is difficult to select hybridization conditions suitable for all probes. However, by using the probes listed in Table 2, multiplex reactions can be performed at a constant temperature with constant stirring speed, because these probes are designed to hybridize to their respective targets under similar hybridization conditions. In some embodiments, the hybridization temperature is 35-50°C, 40-50°C, 40-45°C, or 45-50°C. In some embodiments, hybridization is carried out using a thermomixer at a rotation speed of 700 to 1000 rpm, 750 to 1000 rpm, 800 to 1000 rpm, 900 to 1000 rpm, 700 to 750 rpm, 700 to 800 rpm, 750 to 800 rpm, or 800 to 900 rpm.

[0130] The probe-bound product can be detected by detecting a gene-specific primer, a universal primer, or a split probe in the product. Therefore, primers or probes are usually modified to be detectable. They may be modified to have fluorescent or chemiluminescent activity, or to be chromogenic or colorimetric by being directly or indirectly linked to a detectable molecule. In some embodiments, one or both primers in the primer pair are linked to biotin or other compounds capable of binding to a detectable molecule conjugated to streptavidin with a signal. The detectable signal may be a dye, a chemiluminescent dye, a fluorescent molecule such as phycoerythrin (PE) or cyanine, a radioisotope, a spin label, a hapten, a quantum dot, a bead, aminohexyl, pyrene, or an enzyme for a color-developing reaction, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). The enzyme used in the color-developing reaction catalyzes the production of a colored compound in the presence of a color-developing substrate. In some embodiments, split probes that detect specific NTRK fusion types are each linked to their own unique identifiers, allowing multiple NTRK fusion types to be simultaneously detected and distinguished from one another. The unique identifiers may be oligonucleotides with unique sequences, microbeads, or nanoparticles that contain unique barcodes on their surfaces. The barcodes may be geometric patterns that can be read by an optical scanner equipped with a bright-field imaging system. In some embodiments, the microbeads or nanoparticles are magnetic particles. In some embodiments, the microbeads or nanoparticles are made of synthetic polymers.

[0131] The unique identifier may be linked to the probe directly or via a linker. In some embodiments, the unique identifier is linked to the probe by a direct chemical bond, forming a covalent bond therebetween. In some embodiments, the unique identifier is linked to the probe via a polymer linker. In some embodiments, the unique identifier is linked to the probe by hybridization between complementary nucleotide sequences.

[0132] The disclosed methods can be based on several technology platforms capable of performing multiplex reactions, such as microarray plates, gene chips, microbeads, nanoparticles, membranes, or microfluidic devices. In some embodiments, the probes are immobilized at different locations on a microarray plate, gene chip, or membrane, for example, as an array of spots each containing multiple copies of one type of probe. In certain other embodiments, the probes are associated with microbeads (e.g., magnetic microbeads). In still other embodiments, the probes are coated on a substrate plate of a microfluidic device, with different probes located in different regions of the substrate plate.

[0133] When the probes are immobilized on a DNA microarray plate, the microarray plate can further include a set of control spots, each containing multiple copies of a control probe. The control probe binds to the DNA of housekeeping genes such as β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β2-microglobulin. Therefore, the control spots can be used as internal standards to verify assay performance. Furthermore, the microarray plate can further include a set of anchor spots, each containing multiple copies of an anchor probe. The anchor probe is designed to be detected regardless of the amplification product. Therefore, the anchor spots can be used as position indicators for nearby spots on the microarray plate.

[0134] The present disclosure also provides a method of treating a subject, the method comprising: (a) determining whether a subject is at risk for cancer or a genotype, comprising detecting DNA fragment ligation events in a sample from the subject by the method described in the preceding paragraph, and / or distinguishing alternative splicing events in a sample from the subject by the method described in the preceding paragraph; and (b) administering: (i) a therapeutically effective amount of an siRNA targeting the DNA fragment ligation event and / or the alternative splicing event; (ii) a therapeutically effective amount of an inhibitor of the fusion protein encoded by said DNA fragment ligation event and / or said alternative splicing event; (iii) a therapeutically effective amount of an agent that inhibits the DNA fragment ligation event and / or the fusion protein encoded by the alternative splicing event; (iv) a therapeutically effective amount of an anti-cancer agent selected from the group consisting of cytokines, apoptosis inducers, anti-angiogenic agents, chemotherapeutic agents, radiotherapeutic agents, and anti-cancer immunotoxins; or (v) providing a targeted genome editing treatment in a cell of said subject; Includes:

[0135] In some embodiments, the DNA fragment ligation event and / or the alternative splicing event occurs with the sequence of a partner gene described in the preceding paragraph. In some embodiments, the DNA fragment ligation event and / or the alternative splicing event occurs with the sequence of a target gene described in the preceding paragraph.

[0136] In some embodiments, the alternative splicing event is selected from the group consisting of constitutive splicing, exon skipping, intron retention, mutually exclusive exons, and alternative 5' or 3' splice sites.

[0137] In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, lymphoma, leukemia, or myeloma.

[0138] In some embodiments, the cancer is selected from the group consisting of brain cancer, breast cancer, colon cancer, endocrine cancer, esophageal cancer, female reproductive cancer, head and neck cancer, hepatobiliary cancer, kidney cancer, lung cancer, mesenchymal cell neoplasm, prostate cancer, skin cancer, gastric cancer, exocrine pancreatic tumors, and urinary system cancer.

[0139] In some embodiments, if an NTRK gene fusion (DNA fragment linkage) is detected in a sample from a cancer patient (subject), the patient is predicted to respond to a TRK inhibitor, particularly an NTRK inhibitor such as larotrectinib, entrectinib, LOXO-195, or TPX-0005.

[0140] In some embodiments, the methods disclosed in the preceding paragraphs can be used in prospective analysis of the course or treatment of RNA splicing-related diseases or cancer diseases (see, e.g., Scotti, M., Swanson, "M. RNA mis-splicing in disease," Nat Rev Genet 17, 19-32 (2016); Kim, HK, Pham, MHC, Ko, KS et al. "Alternative splicing isoforms in health and disease," Pflugers Arch-Eur J Physiol 470, 995-1016 (2018); Wang, E. & Aifantis, I. "RNA Splicing and Cancer," Trends in Cancer 6, 631-644 (2020), the contents of which are incorporated herein by reference).

[0141] The present disclosure also provides a kit for detecting a sample having a DNA fragment ligation event and / or an alternative splicing event, the kit comprising: (a) a set of oligonucleotides; (b) a split probe having: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to another DNA fragment, wherein the gaps between the target sites of the split probes on the target nucleic acid to be amplified are within a distance of 0 to 80 bp from each other; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gaps between the target sites of the split probes on the target nucleic acid are within a distance of 0 to 80 bp from each other; and (c) a probe hybridization assay for detecting hybridization signals of split probes, wherein the probe hybridization assay comprises a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, an aminohexyl, and a pyrene; Includes:

[0142] In some embodiments, the kit includes the set of oligonucleotides that are gene-specific primers or gene-specific probes. In some embodiments, the kit includes at least two pairs of gene-specific primers described in the preceding paragraph. In some embodiments, the gene-specific primers are designed to obtain a target nucleic acid from a partner DNA fragment as an upstream DNA fragment. In some embodiments, the gene-specific primers are designed to obtain a target nucleic acid from a partner DNA fragment as a downstream DNA fragment.

[0143] In some embodiments, the kit further comprises a universal primer as described in the preceding paragraph.

[0144] In some embodiments, at least one of the gene-specific primers targets a DNA fragment junction boundary.

[0145] In some embodiments, the gene-specific primers are targeted within a distance of 0-80 bp from the DNA fragment junction boundary.

[0146] In some embodiments, the first split probe and the second split probe are targeted within a distance of 0-40 bp from the DNA fragment junction boundary.

[0147] In some embodiments, the first split probe is complementary to a partner DNA fragment having the sequence of a partner gene described in the preceding paragraph and any complementary sequences thereof. In certain other embodiments, the second split probe is complementary to a target DNA fragment having the sequence of a target gene described in the preceding paragraph and any complementary sequences thereof. In certain other embodiments, the third split probe is complementary to a third DNA fragment having the sequence of a partner gene or target gene described in the preceding paragraph and any complementary sequences thereof.

[0148] In some embodiments, the first split probe is complementary to a partner DNA fragment comprising the sequence of a partner gene described in the preceding paragraph and any complementary sequences thereof. In some embodiments, the second split probe is complementary to a target DNA fragment comprising the sequence of a target gene described in the preceding paragraph and any complementary sequences thereof. In some embodiments, the third split probe is complementary to a third DNA fragment comprising the sequence of a partner gene or target gene described in the preceding paragraph and any complementary sequences thereof.

[0149] In some embodiments, the split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36 and the complement of any of them.

[0150] In some embodiments, one or both primers in the primer pair are linked to biotin or other compounds capable of binding, with a signal, to a detectable molecule conjugated to streptavidin.

[0151] In some embodiments, the split probes are 10-60 bp in length.

[0152] In some embodiments, the amplified target nucleic acid is 200 bp or less in length.

[0153] In some embodiments, probe hybridization assays are designed for use with a variety of detectable signals, such as dyes, chemiluminescent dyes, fluorescent molecules such as phycoerythrin (PE) or cyanines, radioisotopes, spin labels, haptens, quantum dots, beads, aminohexyl, pyrene, or enzymes that detect colorimetric reactions such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

[0154] In some embodiments, split probes for detecting specific NTRK fusion types are designed with their own unique identifiers linked to them. The unique identifiers may be oligonucleotides with unique sequences, microbeads, or nanoparticles with unique barcodes on their surfaces. The barcodes may be geometric patterns that can be read by an optical scanner equipped with a bright-field imaging system. In some embodiments, the microbeads or nanoparticles are magnetic particles. In some embodiments, the microbeads or nanoparticles are made of synthetic polymers.

[0155] In some preferred embodiments, the kit includes a universal primer, and when the NTRK fusion-specific primer pair is used in combination with a universal primer, the NTRK fusion-specific forward primer in each pair of the NTRK fusion-specific primers further comprises the nucleotide sequence of the universal forward primer in the universal primer pair, and the NTRK fusion-specific reverse primer in each pair of the NTRK fusion-specific primers further comprises the nucleotide sequence of the universal reverse primer in the universal primer pair.

[0156] In some embodiments, the kit further comprises a reverse transcriptase for reverse transcribing RNA isolated from the sample, and also comprises a DNA polymerase for amplifying cDNA produced by the reverse transcription.

[0157] In some embodiments, the kit further includes an internal standard. The internal standard may be a positive control sample containing an NTRK gene fusion or a negative control sample lacking an NTRK gene fusion. In some embodiments, the internal standard is an FFPE tissue section, peripheral blood mononuclear cells (PBMCs), blood, plasma, other cells or body fluids, a nucleic acid, or an oligonucleotide.

[0158] The kit described in the preceding paragraph takes advantage of the detection accuracy in finding DNA fragment ligation and alternative splicing events, resulting in a reliable analysis of clinical genotypes.

[0159] The present disclosure is further illustrated by the following examples, which are offered by way of illustration and not by way of limitation. [Example]

[0160] Example 1

[0161] Detection of MET gene mutations by a one-step PCR target-probe hybridization assay

[0162] The one-step PCR target-probe hybridization assay can simultaneously detect possible MET alternative splice forms in a single reaction. Figure 1 shows the overall process of this assay. This process includes the steps of obtaining RNA from a sample, reverse transcribing the RNA to obtain cDNA, PCR-amplifying the MET alternative splice region of the cDNA (i.e., the target cDNA) using multiple MET mutation-specific primer pairs to obtain an amplification product of the target cDNA, PCR-amplifying this primary amplification product using a universal primer pair to obtain a secondary amplification product of the target cDNA, probe hybridization with the amplified target cDNA using a split probe, and detection of the probe-bound product. The following is an example of this assay.

[0163] Preparation of split probes

[0164] Prior to the assay, probes targeting MET exon 14 skipping mutations are designed based on the nucleotide sequence of the fusion region in the RNA transcript of the MET gene (Table 3). The sequences in Table 3 are for the 5' partner (exon 13 of the MET gene) and the 3' target (exon 15 of the MET gene), respectively. Split probes (e.g., those shown in Table 1) are designed to bind to the sequences listed in Table 3. The split probes are immobilized on a microarray plate in the form of an array of spots, where each spot contains multiple copies of one type of probe.

[0165] [Table 3]

[0166] PCR enrichment using MET mutation-specific primer pairs

[0167] To surrogate for clinical samples with MET alternative splicing, IDT synthesized an oligonucleotide used as a positive standard template. This MET alternative splicing oligo was amplified by PCR using the MET mutation-specific primer pair shown in Table 4. This primer pair, which can bind to the 5' and 3' ends of the MET alternative splicing oligo, was synthesized by IDT. The reverse primer of the primer pair was modified with biotin at the 5' end for subsequent interaction with streptavidin-phycoerythrin (SA-PE) conjugate (Thermo Fisher Scientific). PCR was performed using Platinum Taq DNA polymerase High Fidelity (Thermo Fisher Scientific) according to the manufacturer's instructions for 30 thermal cycles in a Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific).

[0168] [Table 4]

[0169] Probe Hybridization and Signal Detection

[0170] The MET alternative splicing oligo amplification products are transferred to pre-blocked wells for hybridization. Each well is then printed with an array of split probe spots, including a spot for a MET alternative splicing-specific probe (e.g., those listed in Table 1), a spot for a control probe, and a spot for an anchor probe. After hybridization, a fluorescent SA-PE conjugate is added to the well and binds to the biotin in the amplification product, generating a colored product where the probe-target hybrid exists. By photographing the wells with a specialized camera and identifying the location of the colored spots within the wells, specific hybridization, indicating the presence of specific MET alternative splicing, can be determined. The locations of the colored spots can be analyzed by computer.

[0171] Example 2

[0172] Detection of NTRK gene mutations by a two-step PCR target-probe hybridization assay

[0173] The two-step PCR target-probe hybridization assay is another method designed to simultaneously detect multiple potential NTRK fusions in a single reaction. Figure 2 shows the overall process of this assay, including the steps of obtaining RNA from a sample, reverse transcribing the RNA to obtain cDNA, PCR-amplifying the NTRK fusion region of the cDNA (i.e., target cDNA) using multiple NTRK fusion-specific primer pairs to obtain a primary amplification product of the target cDNA, PCR-amplifying the primary amplification product using a universal primer pair to obtain a secondary amplification product of the target cDNA, subjecting the amplified target cDNA to a probe hybridization assay, and detecting the probe-bound product. The following is an example of this assay.

[0174] RNA extraction and reverse transcription

[0175] Both DNA and RNA were extracted from FFPE tissue specimens from cancer patients using the RecoverAll Total Nucleic Acid Isolation Kit (Cat No: AM1975, Ambient Technologies) according to the manufacturer's instructions. Reverse transcription of 100 ng of total RNA was performed at 42°C for 30–60 min using the SuperScript cDNA Synthesis Kit (Cat No: 11754050, Invitrogen) and random hexanucleotide primers. This step yielded 10 μL of cDNA product.

[0176] PCR enrichment using NTRK fusion-specific primer pairs

[0177] Each primer in the NTRK fusion-specific primer pair used in this assay is designed to have two segments. One segment, called the fusion-specific segment, is used to bind to the 5' or 3' end of the fusion sequence of a specific NTRK fusion. The other segment, called the universal segment, contains the nucleotide sequence of a universal primer used in the second round of PCR. The universal segment is always located upstream, i.e., 5', of the fusion-specific segment (Figure 2). The universal primer can be any of the primers listed in Table 5, and each universal primer can be used as both the universal forward primer and the universal reverse primer. Table 6 lists the fusion-specific segments of several fusion-specific primer pairs used in this assay.

[0178] [Table 5]

[0179] [Table 6]

[0180] For fusion-specific PCR, 7 μL of water was added to 10 μL of cDNA product, and the resulting mixture (17 μL) was divided into four equal pools of 4 μL each, leaving 1 μL as dead volume. The number of pools was determined based on primer performance. More specifically, the primer efficiency of each fusion-specific primer pair was first measured, and fusion-specific primer pairs with similar efficiencies were mixed to form a single primer pool, resulting in a total of four primer pools (designated P1, P2, P3, and P4). Each primer pool contained 23–48 fusion-specific primers (Table 7) and was added to one pool of cDNA (4 μL). The cDNA in each pool was then subjected to Veritigen PCR over 25 thermal cycles using a multiplex PCR kit (Cat No: 206143, Qiagen) according to the manufacturer's instructions. TM Amplification was performed using a 96-well Thermal Cycler (Thermo Fisher Scientific) to obtain 10 μL of the first amplification product, i.e., four multiplex PCR reactions were performed to obtain four pools of the first amplification product.

[0181] [Table 7]

[0182] PCR enrichment by using universal primer pairs

[0183] Because each fusion-specific primer contains the nucleotide sequence of a universal primer at its 5' end, the first amplification product can be further amplified by PCR using a universal primer pair comprising a universal forward primer having a sequence selected from SEQ ID NOS: 40-49 and a universal reverse primer having a sequence selected from SEQ ID NOS: 40-49. The universal reverse primer is biotinylated. For the second round of PCR, each of the four pools of first amplification products is diluted 100-fold into the final reaction mix and amplified using Platinum SuperFi II PCR Master Mix (Cat No: 12368010, Invitrogen) according to the manufacturer's instructions for 25 thermal cycles in a Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific) to obtain 10 μL of second amplification product. That is, four PCR reactions are performed to obtain four pools of second amplification product.

[0184] Probe Hybridization and Signal Detection

[0185] Four pools of the second amplification products (40 μL in total) were combined, and 18 μL of the resulting pool was mixed with 3 μL of water to obtain a mixture. This mixture was placed in a 96-well PCR plate (Cat No: P46-4TI-1000 / c, 4-titude). The second amplification products were denatured at 96°C for 5 minutes and transferred to pre-blocked wells, each of which was pre-printed with an array of probe spots, including a split probe spot, nine control probe spots, and ten anchor probe spots. The split probes were selected from SEQ ID NOs: 32, 33, 35, and 36 (Table 2). Figures 5 and 6 show the distribution of different probes in one well. Target-probe hybridization was performed at 50°C for 15 minutes with shaking. After hybridization, the wells were cooled and washed twice. A buffer containing streptavidin-alkaline phosphatase conjugate is then added to the wells to allow biotin-streptavidin interaction, followed by the addition of a substrate for alkaline phosphatase to form a colored product where the probe-target hybrid is present. Specific hybridization, indicative of the presence of a specific NTRK fusion, is measured by photographing the wells with a camera and identifying the location of the colored spot in the well. The location of the colored spot can then be analyzed by computer.

[0186] Example 3

[0187] Detection of EGFRvIII mutations by a two-step PCR target-probe hybridization assay

[0188] Here, a two-step PCR target-probe hybridization assay is used to detect EGFRvIII mutations. Figure 3 shows the overall process of this assay, which includes the steps of obtaining RNA from a sample, reverse transcribing the RNA to obtain cDNA, PCR-amplifying the EGFRvIII mutation region of the cDNA (i.e., target cDNA) using an EGFRvIII mutation-specific primer pair to obtain a first amplification product of the target cDNA, PCR-amplifying the first amplification product using a universal primer pair to obtain a second amplification product of the target cDNA, hybridizing the amplified target cDNA with a probe, and detecting the probe-bound product. The following is an example of this assay.

[0189] RNA extraction and reverse transcription

[0190] Both DNA and RNA were extracted from FFPE tissue specimens from cancer patients using the RecoverAll Total Nucleic Acid Isolation Kit (Cat. No. AM1975, Ambient Technologies) according to the manufacturer's instructions. 100 ng of total RNA was reverse transcribed using the SuperScript cDNA Synthesis Kit (Cat. No. 11754050, Invitrogen) and random hexanucleotide primers at 42°C for 30–60 min to obtain 10 μL of cDNA product.

[0191] PCR enrichment by using EGFRvIII mutation-specific primer pairs

[0192] Each primer in the EGFRvIII mutation-specific primer pair used in this assay is designed to have two segments. One segment, referred to as the alternative splice-specific segment, is used to bind to the 5' or 3' end of the EGFRvIII mutation sequence. The alternative splice-specific segment may have the sequence of SEQ ID NO: 62 or 63 (Table 8). The other segment, referred to as the universal segment, contains the nucleotide sequence of a universal primer used in the second round of PCR. The universal segment is always located upstream, i.e., 5', relative to the alternative splice-specific segment. The universal primer may be any of the primers listed in Table 5, and each universal primer can be used as both the universal forward primer and the universal reverse primer.

[0193] [Table 8]

[0194] For the alternative splicing-specific PCR, 10 μL of the cDNA product was amplified using a multiplex PCR kit (Cat No: 206143, Qiagen) according to the manufacturer's instructions in a Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific) for 15 to 30 thermal cycles to obtain 10 μL of the first amplification product.

[0195] PCR enrichment by using universal primer pairs

[0196] Because each mutation-specific primer contains the nucleotide sequence of a universal primer at its 5' end, the first amplification product can be further amplified by PCR using a universal primer pair containing a universal forward primer having a sequence selected from SEQ ID NOs: 40 to 49 and a universal reverse primer having a sequence selected from SEQ ID NOs: 40 to 49. The universal reverse primer is biotinylated. For the second round of PCR, the first amplification product is diluted 100-fold in the final reaction mix and amplified using Platinum SuperFi II PCR Master Mix (Cat No: 12368010, Invitrogen) according to the manufacturer's instructions for 15 to 30 thermal cycles in a Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific) to obtain 10 μL of second amplification product.

[0197] Probe Hybridization and Signal Detection

[0198] The second amplification products were placed in a 96-well PCR plate (Cat No: P46-4TI-1000 / C, 4-degree). The second amplification products were denatured at 96°C for 5 minutes and transferred to pre-blocked wells, each pre-printed with an array of probe spots, including 117 split probes, 9 control probes, and 10 anchor probes (Figure 4). Split probes (e.g., those shown in Table 1) were designed to bind to the sequences shown in Table 9. Target-probe hybridization was carried out at approximately 50°C for 15 minutes with shaking. After hybridization, the wells were cooled and washed twice. A buffer containing streptavidin-alkaline phosphatase conjugate was then added to the wells to allow biotin-streptavidin interaction, followed by the addition of a substrate for alkaline phosphatase, resulting in the formation of a colored product where the probe-target hybrids were present. Specific hybridization, indicative of the presence of EGFRvIII, can be measured by photographing the wells with a camera and identifying the location of the color spots in the wells, which can be analyzed by computer.

[0199] [Table 9]

[0200] Example 4

[0201] Analytical sensitivity of gene fusion detection by a two-step PCR target-probe hybridization assay

[0202] Before analysis, split probes targeting NTRK fusions are designed based on the nucleotide sequence of the fusion region in the RNA transcript of the NTRK gene.To examine the analytical sensitivity of the fusion probe assay, DNA templates containing both known and previously unreported NTRK fusions are synthesized (e.g., as shown in Table 1, Table 2, and Table 10).A total of 165 synthetic DNA templates are available for known NTRK fusions, and 50 synthetic DNA templates are available for novel NTRK fusions.To examine the sensitivity of each fusion probe, each template is diluted to 1,000 copies.Based on the detection range of each probe, the signal from the probe must be higher than the signal threshold for that signal to be considered to be within a certain analytical sensitivity.For known NTRK fusions (e.g., as shown in Figure 7) (a total of 165), the data showed that 85% (141) of NTRK fusion transcripts are sensitive at 1,000 copies. For the novel NTRK fusion types (e.g., shown in Figure 8 ), the data showed that there were 98% ( 49 ) NTRK fusion transcripts with sensitivity at 1000 copies.

[0203] [Table 10]

[0204] Example 5

[0205] Clinical sample validation of fusion detection by a two-step PCR target-probe hybridization assay

[0206] To validate the data analysis algorithm, 38 clinical FFPE-derived RNA samples were analyzed on an NGS platform using the ACTFusion panel (ACT Genomics Co., Ltd.). Of the 38 thyroid cancer FFPE samples after data analysis, only one was NTRK fusion-positive, containing the "ETV6-NTRK3" fusion. The NTRK fusion split-probe assay test results are shown in Table 11. These results are consistent with the NGS results using the ACTFusion panel. The NGS assay test results using the ACTFusion panel protocol are shown in Table 12. The remaining 37 thyroid cancer FFPE samples were NTRK fusion-negative, and these samples were also negative in the NGS assay. These results demonstrate 100% agreement between the ACTFusion panel and the split-probe assay for these 38 clinical FFPE samples. The split-probe chip assay performance data calculated using the sample results are shown in Table 13 as NTRK fusion-positive or -negative.

[0207] [Table 11]

[0208] [Table 12]

[0209] [Table 13] Specificity = 100.00% (95% confidence interval: 90.51% to 100.00%) Accuracy = 100.00% (95% confidence interval: 90.75% to 100.00%) Positive agreement rate (PPA) = 100% (95% confidence interval: 2.5% to 100.00%) Positive predictive value (PPV) = 100.00% (95% confidence interval: 2.5% to 100.00%)

[0210] Example 6

[0211] BCR-ABL by two-step PCR target-probe hybridization assay 35INS Mutation detection

[0212] Here, we used a two-step PCR target-probe hybridization assay to identify BCR-ABL 35INS The assay comprises the steps of obtaining RNA from a sample, reverse transcribing the RNA to obtain cDNA, and detecting the BCR-ABL mutation. 35INS The BCR-ABL cDNA (i.e., target cDNA) was cloned using a mutation-specific primer pair. 35INS The methods include the same steps of PCR amplifying the mutation region to obtain a first amplification product of the target cDNA, PCR amplifying the first amplification product of the target cDNA using a universal primer pair to obtain a second amplification product of the target cDNA, hybridizing a probe to the amplified target cDNA, and detecting the probe-bound product.

[0213] BCR-ABL 35INS PCR enrichment using mutation-specific and universal primer pairs

[0214] BCR-ABL used in this assay 35INS Each primer in the mutation-specific primer pair is designed to have two segments. One segment, called the alternative splicing-specific segment, is a BCR-ABL mutation. 35INSThe alternative splice-specific segment may have the sequence of SEQ ID NO: 67 or 68 (Table 14). The other segment, called the universal segment, contains the nucleotide sequence of a universal primer used in the second round of PCR. For alternative splice-specific PCR, the cDNA product is amplified according to the manufacturer's instructions, thereby obtaining a first amplification product and a second amplification product.

[0215] [Table 14]

[0216] Probe Hybridization and Signal Detection

[0217] The second amplification products are denatured at 96°C for 5 minutes and transferred to pre-blocked wells, each of which is printed with an array of probe spots including the split probe spots (e.g., as shown in Table 1), control probe spots, and anchor probe spots. The split probes are designed to bind to the sequences shown in Table 15, and target-probe hybridization and signal detection follow our previous description.

[0218] BCR-ABL 35INS The mutation and enrichment process has been previously reported (Yuda, Junichiro, et al. "Persistent detection of alternatively spliced BCR-ABL variant results in a failure to achieve a deep molecular response." Cancer science 108.11(2017):2204-2212), which are incorporated herein by reference.

[0219] Because BCR-ABL alternative splicing events result in mixed splice forms at the mRNA level, the referenced approaches are often limited in their reliability to operate at a single design point. These approaches have in common that they probe, or are designed to probe, one splice isoform at a time. In contrast, the split-probe assays described above allow for the detection of multiple splice isoforms (e.g., BCR-ABL or BCR-ABL). 35INS ) can be probed, and splice forms can also be simultaneously identified through signal analysis. The efficiency of split-probe assays is enhanced because the same probe can be used for multiple splice forms.

[0220] This provides various advantages as a probing system, such as high precision and accuracy, a wide range of reportable novel mutation combinations, low cost, and ease of genetic manipulation.

[0221] [Table 15] The present disclosure also includes the following aspects. <1> (a) obtaining DNA in a sample or DNA obtained from extracted RNA; (b) enriching the DNA with a set of oligonucleotides to obtain a target nucleic acid; (c) probing the target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; including; and (d) detecting a signal reflecting binding of the split probe to the target nucleic acid; 1. A method for detecting a DNA fragment ligation event, comprising: <2> The oligonucleotides of the set are gene-specific primers or gene-specific probes. <1> The method described below. <3> In step (b), the DNA is amplified by multiplex PCR using at least two pairs of gene-specific primers; <1> The method described below. <4> (e) (i) confirming a signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment through confirmation of a signal based on binding of the first split probe to the 5' end of the partner DNA fragment and / or binding of the second split probe to the 3' end of the target DNA fragment; or (iii) whether the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment through confirming the signal based on the binding of the third split probe to the third DNA fragment and the result of the target nucleic acid from an independent PCR; further comprising determining <3> The method described below. <5> At least two pairs of the gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as an upstream DNA fragment; <3> The method described below. <6> At least two pairs of the gene-specific primers are is designed to obtain the target nucleic acid from the partner DNA fragment of <3> The method described below. <7> at least one of the gene-specific primers targets a DNA fragment junction boundary; <3> The method described below. <8> The gene-specific primer targets within a distance of 0 to 80 bp from the DNA fragment junction boundary. <3> The method described below. <9> The first split probe or the second split probe targets within a distance of 0 to 40 bp from a DNA fragment junction boundary. <1> The method described below. <10> The first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequences thereof; <1> The method described below. <11> The second split probe is selected from the group consisting of SEQ ID NOs: 33, 36, and any complementary sequence thereof; <1> The method described below. <12> The third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequence thereof; <1> The method described below. <13> The length of the split probe is 10 to 60 bp. <1> The method described below. <14> In step (c), the target nucleic acid is probed with a split probe and a single probe targeting a DNA fragment junction boundary. <1> The method described below. <15> Specific DNA fragments are ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, and AKAP1 3, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, AT G7.ATP1B.BAG4.BAIAP2L1.BCAN.BCL6.BCR.BICC1.BRD3.BRD4.BTBD1.C APZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB. CHTOP, CLCN6, CLIP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD REBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DN AJC8, EIF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZ R, FAM131B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FR Y、FUS、GKAP1、GOLGA4、GON4L、GOPC、GRB7、GRHL2、GRIPAP、GSE1、GTF2E2 GTF2IRD1, HACL1, HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK 2. KANK1, KCTD16, KCTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTA P1-4, KTN1, LIPI, LMNA, LMNTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, M AGI3, MBIP, MBNL1, MED1, MEF2D, MET, MIR548F1, MKRN1, MLLT1, MLLT10, M LLT11, MLLT3, MLLT4, MPRIP, MRPL24, MSN, MTSS1, MUC2, MYH9, MYO5A, NAC C2, NAV1, NBPF20, NCOA4, NFASC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTR K3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB EAR1, PGAP3, PHC3, PHF20, PICALM, PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2 , RBPMS, RET, RFWD2, RNF213, ROS1, RRBP1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9 , SHC1, SHKBP1, SIL1, SLC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPECC1L, SPTBN 1, SPTBN2, SQSTM1, SRCIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1; <1> The method described below. <16> the target DNA fragment comprises a sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2; <1> The method described below. <17> the partner DNA fragment and the target DNA fragment each comprise different sequences from the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF; <1> The method described below. <18> The DNA fragment ligation events include ACVR2A-AKT3, AFAP1-NTRK1, AFAP1-NTRK2, AFAP1-RET, AGAP3-BRAF, AGBL4-NTRK2, AGGF1-RAF1, AKAP13-NTRK3, AKAP13-RET, AKAP9-BRAF, AKT3-P2RX5, AKT3-PTPRR, AMOTL2-NTRK1, APIP-FGFR2, ARGLU1-NTRK1, ARHGEF11-NTRK1, ARHGEF2-NTRK1, ATG7-RAF1, ATP1B-NTRK1, AXL-MBIP, BAG4-FGFR1, BAIAP2L1-BRAF, BAIAP2L1-MET, BCAN-NTRK1, BCAN-NTRK2 ... K1, BCL6-RAF1, BCR-ABL, BCR-FGFR1, BCR-JAK2, BCR-NTRK2, BCR-RET, BRD3-NUTM1, BRD4-NUTM1, BTBD1-NTRK3, CAPZA2-MET, CBR4-ERBB4, CCDC6-BRAF, CCDC6-RET, CCDC6-ROS1, CD74 -NRG1, CD74-NRG2, CD74-NTRK1, CD74-ROS1, CDK12-ERBB2, CDK5RAP2-BRAF, CEL-NTRK1, CEP170-AKT3, CHTOP-NTRK1, CLCN6-RAF1, CLIP1-ALK, CLIP1-ROS1, CLIP2-BRAF, CLIP2-MET, CLTC-ALK, CLTC-ROS1, CNTRL-KIT, COL25A1-ALK, COL25A1-FGFR2, COX5A-NTRK3, CPD-ERBB2, CTRC-NTRK1, CUX1-BRAF, CUX1-FGFR1, CUX1-RET, DCTN1-ALK, DCTN1-MET, DLG1-NTRK3, DNAJC8-ERBB2, EIF3E-RSPO2, EML1-NTRK2, EML4-ALK, EML4-BRAF, EML4-NTRK3, EML4-RET, EPHB2-NTRK1, EPS15-BRAF, E PS15-MET、EPS15-NTRK1、ERBB2-CDK12、ERBB2-CFB、ERBB2-CNIH4、ERBB2-CTTN、ERBB2-DNAJC7、ERBB2-ENO1、ERBB2-FCGRT、ERBB2-FKBP10、ERBB2-GRB 7、ERBB2-GSE1、ERBB2-GTF2E2 / SMIM18、ERBB2-IKZF3、ERBB2-KRT20、ERBB2-KRT39、ERBB2-KRTAP1-4、ERBB2-LMNTD1、ERBB2-LTBP4、ERBB2-MAD2L2、ERB B2-MED1、ERBB2-PARN、ERBB2-PGAP3、ERBB2-POLD4、ERBB2-PPP1R1B、ERBB2-PRDX4、ERBB2-PSMB3、ERBB2-SHKBP1、ERBB2-SLC39A11、ERBB2-SPTBN2、ER BB2-SRCIN1、ERBB2-TADA2A、ERBB2-TATDN1、ERBB2-XBP1、ERBB2-ZAN、ERBB4-AKAP6、ERBB4-FUS、ERBB4-IKZF2、ERBB4-STK11IP、ERC1-BRAF、ERC1-RET 、ERC1-ROS1、ESRP1-RAF1、ESR1-CCDC170、ETV6-FGFR3、ETV6-NTRK2、ETV6-NTRK3、ETV6-PDGFRB、ETV6-PRDM16、EZR-ERBB4、EZR-ROS1、FAM131B-BRAF、 FAT1-NTRK3、FGFR2-BICC1、FGFR2-TACC3、FGFR3-TACC3、FIP1L1-PDGFRA、FN1-ALK、FN1-ERBB4、FN1-FGFR1、FNDC3B-PIK3CA、FRY-NTRK3、GKAP1-NTRK2、GOLGA4-RAF1、GON4L-NTRK1、GOPC-ROS1、GRHL2-RSPO2、GRIPAP-NTRK1、GTF2IRD1-ALK、HACL1-RAF1、HIP1-ALK、HNRNPA2B1-NTRK3、IKZF2-ERBB4、IQSE C1-RAF1、IRF2BP2-NTRK1、KANK1-NTRK2、KCTD16-NTRK2、KCTD8-NTRK2、KHDRBS1-NTRK3、KIAA1549-BRAF、KIF5B-ALK、KIF5B-RET、KIF5B-ERBB4、KIT-AN KRD11、KIT-PDGFRA、KIT-SLC4A4、KMT2A-AFF1、KMT2A-CREBBP、KMT2A-DAB2IP、KMT2A-ELL、KMT2A-EPS15、KMT2A-MLLT1、KMT2A-MLLT10、KMT2A-MLLT11、 KMT2A-MLLT3、KMT2A-MLLT4、KMT2A-SEP6、KMT2A-SEP9、KTN1-ALK、KTN1-RET、LIPI-NTRK1、LMNA-ALK、LMNA-NTRK1、LMNA-RAF1、LRRC71-NTRK1、LRRFIP1 -FGFR1、LRRFIP1-MET、LYN-NTRK3、MAGI3-AKT3、MBNL1-RAF1、MEF2D-NTRK1、MET-MET、MIR548F1-NTRK1、MKRN1-BRAF、MPRIP-ALK、MPRIP-NTRK1、MPRIP -RAF1、MPRIP-RET、MRPL24-NTRK1、MSN-ALK、MSN-ROS1、MTSS1-ERBB2、MUC2-NTRK2、MYH9-ALK、MYO5A-NTRK3、MYO5A-ROS1、NACC2-NTRK2、NAV1-NTRK2、N BPF20-NTRK2、NCOA4-RET、NFASC-NTRK1、NOS1AP-NTRK1、NOS1AP-NTRK2、NRG2-CYSTM1、NRG2-UBE2D2、NRIP1-RSPO2、P2RY8-NTRK1、PAIP1-NTRK2、PAN3- NTRK2、PAPD7-RAF1、PDE4DIP-NTRK1、PEAR1-NTRK1、PHF20-NTRK1、PICALM-BRAF、PICALM-RET、PLEKHA6-NTRK1、PML-RARA、PPFIBP1-ALK、PPFIBP1-MET、PPFIBP1-、 ROS1、PPL-NTRK1、PRDX1-NTRK1、PRKAR1A-ALK、PRKAR1A-RET、PRKAR1B-ALK、PRKAR1B-BRAF、PRKAR2A-NTRK2、PRPSAP1-NTRK3、PTPRZ1-MET、QKI-NTRK2 、QKI-RAF1、RAC1-AKT3、RAF1-ACTR2、RAF1-AGGF1、RAF1-DAZL、RAF1-ESRP1、RAF1-PHC3、RAF1-TMEM40、RAF1-TRAK1、RAF1-ZPR1、RALGPS2-NTRK3、RANB P2-ALK、RANBP2-FGFR1、RBPMS-NTRK3、RFWD2-NTRK1、RNF213-ALK、RNF213-NTRK1、RRBP1-ALK、RRBP1-RET、SATB1-ALK、SATB1-RET、SCAF11-PDGFRA、SC P2-NTRK1、SCYL3-NTRK1、SDC4-NRG1、SDC4-ROS1、SEC31A-ALK、SHC1-ERBB2、SIL1-NRG2、SLC34A2-MET、SLC34A2-ROS1、SLC45A3-BRAF、SLC45A3-ERG、SLC34A2-MET LC45A3-FGFR2, SLMAP-NTRK2, SND1-BRAF, SPECC1L-NTRK2, SPECC1L-NTRK3, SPTBN1-ALK, SQSTM1-ALK, SQSTM1-FGFR1, SQSTM1-NTRK1, SQSTM1-NTRK2, SQSTM1-NTRK3, SRGAP3-RAF1, SRGAP3-SRGAP3-RAF1, SSBP2-NTRK1, STRN-ALK, STRN-NTRK2, STRN-NTRK3, STRN3-BRAF, STRN3-NTRK1, STRN3-NTRK2, ST RN3-NTRK3、TBC1D2-NTRK2、TBL1XR1-NRG1、TBL1XR1-PIK3CA、TBL1XR1-RET、TFG-ALK、TFG-MET、TFG-NTRK1、TFG-NTRK3、TFG-RET、TFG-ROS1、TIMP3-AL K、TIMP3-NTRK1、TKT-ERBB2、TLE4-NTRK2、TMEM106B-BRAF、TMEM106B-ROS1、TMPRSS2-ERG、TMPRSS2-ETV1、TMPRSS2-ETV4、TMPRSS2-ETV5、TNS3-NTRK2、TP53-NTRK1, TPM3-ALK, TPM3-NTRK1, TPM3-ROS1, TPM4-ALK, TPM4-NTRK3, TPR-ALK, TPR-BRAF, TPR-FGFR1, TPR-MET, TPR-NTRK1, TR AF2-NTRK2, TRAK1-RAF1, TRIM24-BRAF, TRIM24-FGFR1, TRIM24-NTRK2, TRIM24-RET, TRIM33-RET, TRIM33-NTRK1, TRIM4-BRAF, TRI M4-MET, TRIM63-NTRK1, UBE2R2-NTRK3, UFD1-NTRK2, USP13-PIK3CA, VANGL2-NTRK1, VCAN-NTRK2, VCL-ALK, VCL-NTRK2, VIM-NTRK3, VPS18-NTRK3, WHSC1L1-FGFR1, WHSC1L1-NUTM1, WIPF2-ERBB2, WNK2-NTRK2, ZBTB7B-NTRK1 and ZNF710-NTRK3 mutations, <1> The method according to <19> the third DNA fragment comprises a sequence of a partner gene or a target gene; <1> The method described below. <20> In step (b), the DNA is first amplified using gene-specific primers and then amplified using universal primers to obtain the target nucleic acid; <1> The method described below. <21> the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, aminohexyl, and pyrene; <1> The method described below. <22> (a) probing a target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; having; (b) detecting a signal reflecting binding of the split probe to the target nucleic acid; (c) (i) confirming a signal based on the binding of the first split probe to the 3' end of the partner DNA fragment and / or the binding of the second split probe to the 5' end of the target DNA fragment, that the partner DNA fragment is an upstream DNA fragment and / or the target DNA fragment is a downstream DNA fragment; (ii) determining that the partner DNA fragment is a downstream DNA fragment and / or the target DNA fragment is an upstream DNA fragment through confirmation of a signal based on the binding of the first split probe to the 5' end of the partner DNA fragment and / or the binding of the second split probe to the 3' end of the target DNA fragment; or (iii) the third DNA fragment linked to the partner DNA fragment and the target DNA fragment by confirming a signal based on the binding of the third split probe to the third DNA fragment; To determine; and (d) comparing whether the length of the target nucleic acid is identical to a reference sequence; A method for distinguishing alternative splicing events, comprising: <23> the target nucleic acid is amplified using a set of oligonucleotides; <22> The method described below. <24> The target nucleic acid is amplified by multiplex PCR using at least two pairs of gene-specific primers; <22> The method described below. <25> (e) reconfirmation by independent PCR; further comprising: <24> The method described below. <26> At least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as an upstream DNA fragment; <24> The method described below. <27> At least two pairs of gene-specific primers are designed to obtain the target nucleic acid from the partner DNA fragment as a downstream DNA fragment; <24> The method described below. <28> At least one of the gene-specific primers is Targeting <24> The method described below. <29> The gene-specific primer targets within a distance of 0 to 80 bp from the DNA fragment junction boundary. <24> The method described below. <30> The product of the multiplex PCR is then amplified using universal primers to obtain the target nucleic acid. <24> The method described below. <31> the distance between the target sites of the first and second split probes and the DNA fragment ligation boundary is within the range of 0 to 40 bp; <22> The method described below. <32> The length of the split probe is 10 to 60 bp. <22> The method described below. <33> In step (a), the target nucleic acid is probed with a split probe and a single probe targeting a DNA fragment junction boundary; <22> The method described below. <34> the partner DNA fragment and the target DNA fragment each comprise different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF; <22> The method described below. <35> the alternative splicing event is a BCR-ABL mutation. <22> The method described below. <36> the third DNA fragment comprises a sequence of a partner gene or a target gene; <22> The method described below. <37> the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, aminohexyl, and pyrene; <22> The method described below. <38> (a) In a sample from a subject, <1> and / or detecting a DNA fragment ligation event by the method described in <22> determining whether the subject is at risk for cancer or a genotype, comprising distinguishing alternative splicing events by the method described in (b) administering: (i) a therapeutically effective amount of an siRNA targeting the DNA fragment ligation event and / or the alternative splicing event; (ii) a therapeutically effective amount of an inhibitor of the fusion protein encoded by said DNA fragment ligation event and / or said alternative splicing event; (iii) a therapeutically effective amount of an agent that inhibits the DNA fragment ligation event and / or the fusion protein encoded by the alternative splicing event; (iv) a therapeutically effective amount of an anti-cancer agent selected from the group consisting of cytokines, apoptosis inducers, anti-angiogenic agents, chemotherapeutic agents, radiotherapeutic agents, and anti-cancer immunotoxins; or (v) providing a targeted genome editing treatment in a cell of said subject; A method of treating a subject, comprising: <39> Recombinant DNA fragments are ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, AKAP13. AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG7, AT P1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, CAPZA2 BR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CHTOP, CLCN 6. CLIP1, CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD, CREBBP, CTRC TTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, EIF3E, ELL. EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131B, FAT1, FC GRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1, GOLGA4, G ON4L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL1, HIP1, HN RNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16, KCTD8, KHDR BS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LMNTD1 LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1, MEF2D, MET 、MIR548F1、MKRN1、MLLT1、MLLT10、MLLT11、MLLT3、MLLT4、MPRIP、MRPL24、M SN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV1, NBPF20, NCOA4, NFASC, NOS1AP, N RG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PARN DE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM, PLEKHA6, PML.POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PRDX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF213, ROS1, RRB P1, SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SHKBP1, SIL1, SLC34A2, S LC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPECC1L, SPTBN1, SPTBN2, SQSTM1, SRCIN1 , SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1; <38> The method according to <40> The ligation event of the DNA fragments is selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2. appears together with the sequence of the target gene to be <38> The method described below. <41> the alternative splicing events appear with different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF; <38> The method described below. <42> the DNA fragment ligation event or the alternative splicing event is a BCR-ABL mutation; <38> The method described below. <43> The alternative splicing event is selected from the group consisting of constitutive splicing, exon skipping, intron retention, mutually exclusive exons, and alternative 5' or 3' splice sites. <38> The method described below. <44> the cancer is selected from the group consisting of carcinoma, sarcoma, lymphoma, leukemia, and myeloma; <38> The method described below. <45> the cancer is selected from the group consisting of brain cancer, breast cancer, colon cancer, endocrine cancer, esophageal cancer, female reproductive cancer, head and neck cancer, hepatobiliary cancer, kidney cancer, lung cancer, mesenchymal cell neoplasm, prostate cancer, skin cancer, gastric cancer, exocrine pancreatic tumor, and urinary system cancer; <38> The method described below. <46> (a) a set of oligonucleotides; (b) Split probes, including: (i) a first split probe complementary to the 3' end of a partner DNA fragment, a second split probe complementary to the 5' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of a partner DNA fragment, a second split probe complementary to the 3' end of a target DNA fragment, and / or a third split probe complementary to a third DNA fragment, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; and (c) a probe hybridization assay for detecting hybridization signals of split probes, wherein the probe hybridization assay comprises a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, an aminohexyl, and a pyrene; 1. A kit for detecting a sample having a DNA fragment ligation event and / or an alternative splicing event, comprising: <47> The oligonucleotides of the set are gene-specific primers or gene-specific probes. <46> The kit according to claim 1. <48> containing at least two pairs of gene-specific primers, <46> The kit according to claim 1. <49> the gene-specific primer is designed to obtain the target nucleic acid from the partner DNA fragment as an upstream DNA fragment; <48> The kit according to claim 1. <50> the gene-specific primer is designed to obtain the target nucleic acid from the partner DNA fragment as a downstream DNA fragment; <48> The kit according to claim 1. <51> further comprising a universal primer, <46> The kit according to claim 1. <52> at least one of the gene-specific primers targets a DNA fragment junction boundary; <48> The kit according to claim 1. <53> The gene-specific primer targets within a distance of 0 to 80 bp from the DNA fragment junction boundary. <48> The kit according to claim 1. <54> The first split probe or the second split probe targets within a distance of 0 to 40 bp from a DNA fragment junction boundary. <46> The kit according to claim 1. <55> The first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequences thereof; <46> The kit according to claim 1. <56> The second split probe is selected from the group consisting of SEQ ID NOs: 33, 36, and any complementary sequence thereof; <46> The kit according to claim 1. <57> The third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequence thereof; <46> The kit according to claim 1. <58> The length of the split probe is 10 to 60 bp. <46> The kit according to claim 1. <59> further comprising a single probe targeting a DNA fragment junction boundary, <46> The kit according to claim 1. <60> ACVR2A, AFAP1, AFF1, AGAP3, AGBL4, AGGF1, AKAP13, AKAP6, AKAP9, AMOTL2, ANKRD1 1, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG7, ATP1B, BAG4, BAIAP2L1, BCAN, BCL6, BCR, BICC1, BRD3, and BRD4 BTBD1, CAPZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CHTOP, CLCN6, CLIP1 CLIP2, CLTC, CNIH4, CNTRL, COL25A1, COX5A, CPD, CREBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DC TN1, DLG1, DNAJC7, DNAJC8, EIF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM13 1B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDC3B, FRY, FUS, GKAP1, GOLGA4, GON4L, GOPC, GR B7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL1, HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2. JAK2, KANK1, KCTD16, KCTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LM NTD1, LRRC71, LRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1, MEF2D, MET, MIR548F1, MKRN1, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MP RIP, MRPL24, MSN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV1, NBPF20, NCOA4, NFASC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAI P1, PAN3, PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM, PLEKHA6, PML, POLD4, PPFIBP1, PPL, PPP1R1B, PRDM16, PR DX1, PRDX4, PRKAR1A, PRKAR1B, PRKAR2A, PRPSAP1, PSMB3, PTPRR, PTPRZ1, QKI, RAC1, RALGPS2, RANBP2, RBPMS, RET, RFWD2, RNF213, ROS1, RRBP1 , SATB1, SCAF11, SCP2, SCYL3, SDC4, SEC31A, SEP6, SEP9, SHC1, SHKBP1, SIL1, SLC34A2, SLC39A11, SLC45A3, SLC4A4, SLMAP, SMIM18, SND1, SPEC C1L, SPTBN1, SPTBN2, SQSTM1, SRCIN1, SRGAP3, SSBP2, STK11IP, STRN, STRN3, TACC3, TADA2A, TATDN1, TBC1D2, TBL1XR1, TFG, TIMP3, TKT, TLE4, complementary to the partner DNA fragment comprising a sequence of a partner gene selected from the group consisting of TMEM106B, TMEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1; <46> The kit according to claim 1. <61> the second split probe is complementary to the target DNA fragment comprising a sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2; <46> The kit according to claim 1. <62> the first split probe and the second split probe are complementary to the partner DNA fragment and the target DNA fragment, respectively, containing different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF; <46> The kit according to claim 1. <63> the DNA fragment ligation event or the alternative splicing event is a BCR-ABL mutation; <46> The kit according to claim 1. <64> the third split probe is complementary to the third DNA fragment containing a sequence of a partner gene or a target gene; <46> The kit according to claim 1.

Claims

1. (a) obtaining DNA in a sample or DNA obtained from extracted RNA; (b) enriching the DNA with a set of gene-specific oligonucleotides to obtain a target nucleic acid, wherein the target nucleic acid is a DNA fragment junction sequence comprising a partner DNA fragment and a target DNA fragment; (c) forming separate probe-target hybrids by probing the target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of the partner DNA fragment, a second split probe complementary to the 5' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of the partner DNA fragment, a second split probe complementary to the 3' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; wherein the first split probe, the second split probe, and optionally the third split probe are separately immobilized to a solid support at different locations; and (d) detecting signals at the locations of the individual probe-target hybrids to identify each split probe specific to the partner DNA fragment or the target DNA fragment, wherein: (i) the partner DNA fragment is an upstream DNA fragment and the target DNA fragment is a downstream DNA fragment, or (ii) the partner DNA fragment is a downstream DNA fragment and the target DNA fragment is an upstream DNA fragment; and Optionally, determining whether the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment, and confirming the DNA fragment ligation event through the target nucleic acid result from an independent PCR; 1. A method for detecting a DNA fragment ligation event, comprising:

2. The method of claim 1 , wherein the set of gene-specific oligonucleotides is a gene-specific primer or a gene-specific probe.

3. 2. The method of claim 1, wherein in step (b), the DNA is amplified by multiplex PCR using at least two pairs of gene-specific primers.

4. The method of claim 3 , wherein at least one of the gene-specific primers targets a DNA fragment junction boundary.

5. 4. The method of claim 3, wherein the gene-specific primers target within a distance of 0 to 80 bp from a DNA fragment junction boundary.

6. 2. The method of claim 1, wherein the first split probe or the second split probe targets within a distance of 0 to 40 bp from a DNA fragment junction boundary.

7. 2. The method of claim 1, wherein the first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequence thereof.

8. 2. The method of claim 1, wherein the second split probe is selected from the group consisting of SEQ ID NOs: 33, 36, and any complementary sequence thereof.

9. 2. The method of claim 1, wherein the third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequence thereof.

10. The method of claim 1, wherein the split probe has a length of 10 to 60 bp.

11. 10. The method of claim 1, wherein in step (c), the target nucleic acid is probed with a split probe and a single probe that targets a DNA fragment junction boundary.

12. The partner DNA fragments are ACVR2A, AFAF1, AFF1, AGAP3, AGBL4, AGGF1, AKAP13, AKAP6, AKAP9, AMOTL2, ANKRD11, APIP, ARGLU1, ARHGEF11, ARHGEF2, ATG7, ATP1B, BAG4, BAIAIP2L1, BCAN, BCL6, BCR, BICC1, BRD3, BRD4, BTBD1, CAPZA2, CBR4, CCDC170, CCDC6, CD74, CDK12, CDK5RAP2, CEL, CEP170, CFB, CHTOP, CLCN6, CLIP1, CLIP2, CLTC, CNIH4, CTRL, COL25A1, COX5A, CPD, CREBBP, CTRC, CTTN, CUX1, CYSTM1, DAB2IP, DAZL, DCTN1, DLG1, DNAJC7, DNAJC8, EIF3E, ELL, EML1, EML4, ENO1, EPHB2, EPS15, ERC1, ESRP1, ETV6, EZR, FAM131B, FAT1, FCGRT, FGFR1, FGFR3, FIP1L1, FKBP10, FN1, FNDBC3B, FRY, FUS, GKAP1, GOLGA4, GON4L, GOPC, GRB7, GRHL2, GRIPAP, GSE1, GTF2E2, GTF2IRD1, HACL1, HIP1, HNRNPA2B1, IKZF2, IKZF3, IQSEC1, IRF2BP2, JAK2, KANK1, KCTD16, KCTD8, KHDRBS1, KIAA1549, KIF5B, KRT20, KRT39, KRTAP1-4, KTN1, LIPI, LMNA, LMNT1, LRRRC71, LRRRFIP1, LTBP4, LYN, MAD2L2, MAGI3, MBIP, MBNL1, MED1, MEF2D, MET, MIR548F1, MKRNI, MLLT1, MLLT10, MLLT11, MLLT3, MLLT4, MPRIP, MRPL24, MSN, MTSS1, MUC2, MYH9, MYO5A, NACC2, NAV1, NBPF20, NCOA4, NFASC, NOS1AP, NRG1, NRIP1, NTRK1, NTRK2, NTRK3, P2RX5, P2RY8, PAIP1, PAN3, PAPD7, PARN, PDE4DIP, PDGFRA, PDGFRB, PEAR1, PGAP3, PHC3, PHF20, PICALM, PLEKHA6, PML,.........、.......................... 、. 1.、..||||. opher、MSBBt、PT)T range、PPTPRPPRtt、1QKK |||||、|||||、||||||、||||||、 、. #..、. portSDC¼l¼ll¼C,C¼t rangetSl>SlEP?ESESPPPPPP, Sbro1、SH The #|||、#*||#|、##|#|、、 TTTTBBB?SPTTTBBB? range 、SS between %longffided anglest between S range !?、SRQPB+SBBEBBIP、S TRµSTTTplplplplTTTTCplCpplTTTTTDtDA range、TTTTTTTTf T pl1、TTBBBB1?2、TTTBBBBB111、TTTTTTTT .............................................

2. The method of claim 1, comprising the sequence of a partner gene selected from the group consisting of MEM40, TMPRSS2, TNS3, TP53, TPM3, TPM4, TPR, TRAF2, TRAK1, TRIM24, TRIM33, TRIM4, TRIM63, UBE2D2, UBE2R2, UFD1, USP13, VANGL2, VCAN, VCL, VIM, VPS18, WHSC1L1, WIPF2, WNK2, XBP1, ZAN, ZBTB7B, ZNF710, and ZPR1.

13. 2. The method of claim 1, wherein the target DNA fragment comprises a sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2.

14. 2. The method of claim 1, wherein the partner DNA fragment and the target DNA fragment each comprise different sequences from the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

15. The DNA fragment ligation events are ACVR2A - AKT3, AFAP1 - NTRK1, AFAP1 - NTRK2, AFAP1 - RET, AGAP3 - BRAF, AGBL4 - NTRK2, AGGF1 - RAF1, AKAP13 - NTRK3, AKAP13 - RET, AKAP9 - BRAF, AKT3 - P2RX5, AKT3 - PTPRR, AMOTL2 - NTRK1, APIP - FGFR2, ARGLU1 - NTRK1, ARHGEF11 - NTRK1, ARHGEF2 - NTRK1, ATG7 - RAF1, ATP1B - NTRK1, AXL - MBIP, BAG4 - FGFR1, BAIAP2L1 - BRAF, BAIAP2L1 - MET, BCAN - NTRK1, BCL6 - RAF1, BCR - ABL, BCR - FGFR1, BCR - JAK2, BCR - NTRK2, BCR - RET, BRD3 - NUTM1, BRD4 - NUTM1, BTBD1 - NTRK3, CAPZA2 - MET, CBR4 - ERBB4, CCDDC6 - BRAF, CCDDC6 - RET, CCDDC6 - ROS1, CD74 - NRG1, CD74 - NRG2, CD74 - NTRK1, CD74 - ROS1, CDK12 - ERBB2, CDK5RAP2 - BRAF, CEL - NTRK1, CEP170 - AKT3, CHTOP - NTRK1, CLCN6 - RAF1, CLIP1 - ALK, CLIP1 - ROS1, CLIP2 - BRAF, CLIP2 - MET, CLTC - ALK, CLTC - ROS1, CTRL - KIT, COL25A1 - ALK, COL25A1 - FGFR2, COX5A - NTRK3, CPD - ERBB2, CTRC - NTRK1, CUX1 - BRAF, CUX1 - FGFR1, CUX1 - RET, DCTN1 - ALK, DCTN1 - MET, DLG1 - NTRK3, DNAJC8 - ERBB2, EIF3E - RSPO2, EML1 - NTRK2, EML4 - ALK, EML4 - BRAF, EML4 - NTRK3, EML4 - RET, EPHB2 - NTRK1, EPS15 - BRAF, EPS15 - MET, EPS15 - NTRK1, ERBB2 - CDK12, ERBB2 - CFB, ERBB2 - CNIH4, ERBB2 - CTTN, ERBB2 - DNAJC7, ERBB2 - ENO1, ERBB2 - FCGRT, ERBB2 - FKBP10, ERBB2 - GRB7, ERBB2 - GSE1,<h2 style=";text-align:left;direction:ltr">10 ... 、SYSYSYS14、SYSYSYS14、SYS SHYWHAR4、SYSYSY22、SYSYS 1、SAR2000、SHA2003、SHA2 ROCK4、SHASH20S1K10、SHASH20S 4、SYSHASHY3、SYSYSHAY1、SYS 2SYSYS11、SYSYSYSY2、SYSYSYS FASH1、SHAR2KYS20、SHAR2SYS1 、SYSCH1K1、SYSYSYS4、SYSYS 6、SYSCHYS、SYSYSYS2、SYSYS SYS10、SYSYSYS、SYSYSYS、SYS1 CY1、SYS1SYS1、SYSYS170、S THIS IS YOURS3、SYS6SYS2、SYS6SYS3 、SYS SYSCH、SYS SYS H16、SYS SYS CAM4、SYSYS1、SYSYSYSYSY、SYSYS1 9. SHY3、CHANGE2SYS1、SHY2SYS3、0 HISTORY 3、DIS10100000000000000 TH、DYSYS4、DYSYS1、DYSYS THIS 、CHSYSH3、SHYSH22、SHY1 ROYS1、DY4SYS1、SYSYS1、0 SHYSYSY2、SYSYSYS1、SYSYS 1HYH、HARSH1HYH1、HARHYHYH、HARSH 20000000000000 00000000000000000000000000 / 000 / 000000000000 RO1、DAM200000000000000000000000000000000000000,000,000,000,0 ROSHYSHY2、SHRYSHY22、SHYS1 FASHION3、SAM1544SHASY、SAMFISHSHASH、 ROCKSHASHY、SHOCKSHASHYS4、SHAIRSHASH 11、SPIRITUALSHIPS、SPIRITSSHIPS4、SPIRITS CHEEKS、KSYSYSYSYS、HYSYSYSYSYS、 ROYSHASHY、HYSHASHYS15、HYSHASHY 11、SYSYSCHYS10、SYSYSYSYS11、 FAMOUS LOVE 6、SYSYSYSY、SYSYSYS、SYSYSYS、LIPI-NTRK1、LMNA-ALK、LMNA-NTRK1、LMNA-RAF1、LRRC71-NTRK1、LRRFIP1-FGFR1、LRRFIP1-MET、LYN-NTRK3、MAGI3-AKT3、MBNL1-RAF1、MEF2D-NTRK1、MET-MET、MIR548F1-NTRK1、MKRN1-BRAF、MPRIP-ALK、MPRIP-NTRK1、MPRIP-RAF1、MPRIP-RET、MRPL24-NTRK1、MSN-ALK、MSN-ROS1、MTSS1-ERBB2、MUC2-NTRK2、MYH9-ALK、MYO5A-NTRK3、MYO5A-ROS1、NACC2-NTRK2、NAV1-NTRK2、NBPF20-NTRK2、NCOA4-RET、NFASC-NTRK1、NOS1AP-NTRK1、NOS1AP-NTRK2、NRG2-CYSTM1、NRG2-UBE2D2、NRIP1-RSPO2、P2RY8-NTRK1、PAIP1-NTRK2、PAN3-NTRK2、PAPD7-RAF1、PDE4DIP-NTRK1、PEAR1-NTRK1、PHF20-NTRK1、PICALM-BRAF、PICALM-RET、PLEKHA6-NTRK1、PML-RARA、PPFIBP1-ALK、PPFIBP1-MET、PPFIBP1-ROS1、PPL-NTRK1、PRDX1-NTRK1、PRKAR1A-ALK、PRKAR1A-RET、PRKAR1B-ALK、PRKAR1B-BRAF、PRKAR2A-NTRK2、PRPSAP1-NTRK3、PTPRZ1-MET、QKI-NTRK2、QKI-RAF1、RAC1-AKT3、RAF1-ACTR2、RAF1-AGGF1、RAF1-DAZL、RAF1-ESRP1、RAF1-PHC3、RAF1-TMEM40、RAF1-TRAK1、RAF1-ZPR1、RALGPS2-NTRK3、RANBP2-ALK、RANBP2-FGFR1、RBPMS-NTRK3、RFWD2-NTRK1、RNF213-ALK、RNF213-NTRK1、RRBP1-ALK、RRBP1-RET、SATB1-ALK、SATB1-RET、SCAF11-PDGFRA、SCP2-NTRK1、SCYL3-NTRK1、SDC4-NRG1、SDC4-ROS1、SEC31A-ALK、SHC1-ERBB2、SIL1-、 NRG2, SLC34A2-MET, SLC34A2-ROS1, SLC45A3-BRAF, SLC45A3-ERG, SLC45A3 -FGFR2, SLMAP-NTRK2, SND1-BRAF, SPECC1L-NTRK2, SPECC1L-NTRK3, SPTBN 1-ALK, SQSTM1-ALK, SQSTM1-FGFR1, SQSTM1-NTRK1, SQSTM1-NTRK2, SQSTM1 -NTRK3, SRGAP3-RAF1, SRGAP3-SRGAP3-RAF1, SSBP2-NTRK1, STRN-ALK, STRN -NTRK2, STRN-NTRK3, STRN3-BRAF, STRN3-NTRK1, STRN3-NTRK2, STRN3-NTR K3, TBC1D2-NTRK2, TBL1XR1-NRG1, TBL1XR1-PIK3CA, TBL1XR1-RET, TFG-AL K, TFG-MET, TFG-NTRK1, TFG-NTRK3, TFG-RET, TFG-ROS1, TIMP3-ALK, TIMP3 -NTRK1, TKT-ERBB2, TLE4-NTRK2, TMEM106B-BRAF, TMEM106B-ROS1, TMPRSS2 -ERG, TMPRSS2-ETV1, TMPRSS2-ETV4, TMPRSS2-ETV5, TNS3-NTRK2, TP53-NT RK1, TPM3-ALK, TPM3-NTRK1, TPM3-ROS1, TPM4-ALK, TPM4-NTRK3, TPR-ALK, TPR-BRAF, TPR-FGFR1, TPR-MET, TPR-NTRK1, TRAF2-NTRK2, TRAK1-RAF1, TR IM24-BRAF, TRIM24-FGFR1, TRIM24-NTRK2, TRIM24-RET, TRIM33-RET, TRIM3 3-NTRK1, TRIM4-BRAF, TRIM4-MET, TRIM63-NTRK1, UBE2R2-NTRK3, UFD1-NTRK2, USP13-PIK3CA, VANGL2-NTRK1, VCAN-NTRK2, VCL-ALK, VCL-NTRK2, VIM-NTRK3, VPS18-NTRK3, WHSC1L1-FGFR1, WHSC1L1-NUTM1, WIPF2-ERBB2, WNK2-NTRK2, ZBTB7B-NTRK1 and ZNF710-NTRK3 mutations.

16. The method of claim 1 , wherein the third DNA fragment comprises a sequence of a partner gene or a target gene.

17. 2. The method of claim 1, wherein in step (b), the DNA is first amplified using gene-specific primers and then amplified using universal primers to obtain the target nucleic acid.

18. 10. The method of claim 1, wherein the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, aminohexyl, and pyrene.

19. (a) obtaining DNA in a sample or DNA obtained from extracted RNA; (b) enriching the DNA with a set of gene-specific oligonucleotides to obtain a target nucleic acid, wherein the target nucleic acid is a DNA fragment junction sequence comprising a partner DNA fragment and a target DNA fragment; (c) forming separate probe-target hybrids by probing the target nucleic acid with a split probe, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of the partner DNA fragment, a second split probe complementary to the 5' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split complementary to the 5' end of the partner DNA fragment; a probe, a second split probe complementary to the 3' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; wherein the first split probe, the second split probe, and optionally the third split probe are separately immobilized on a solid support at different locations; (d) detecting signals at the locations of the individual probe-target hybrids to identify each split probe specific to the partner DNA fragment or the target DNA fragment, wherein: (i) the partner DNA fragment is an upstream DNA fragment and the target DNA fragment is a downstream DNA fragment; or (ii) the partner DNA fragment is a downstream DNA fragment and the target DNA fragment is an upstream DNA fragment; and Optionally, (iii) the third DNA fragment is linked to the partner DNA fragment and the target DNA fragment; and (e) comparing whether the length of the target nucleic acid is identical to that of a reference sequence; A method for distinguishing alternative splicing events, comprising:

20. 20. The method of claim 19, wherein the target nucleic acid is amplified by multiplex PCR using at least two pairs of gene-specific primers.

21. In step (e), reconfirming said alternative splicing event by independent PCR; 21. The method of claim 20, further comprising:

22. 21. The method of claim 20, wherein at least one of the gene-specific primers targets a DNA fragment junction boundary.

23. 21. The method of claim 20, wherein the gene-specific primers target within a distance of 0 to 80 bp from a DNA fragment junction boundary.

24. 21. The method of claim 20, wherein the multiplex PCR products are then amplified using universal primers to obtain the target nucleic acid.

25. 20. The method of claim 19, wherein the distance between the target sites of the first and second split probes and the DNA fragment junction boundaries is in the range of 0 to 40 bp.

26. 20. The method of claim 19, wherein the split probes are 10 to 60 bp in length.

27. 20. The method of claim 19, wherein in step (c), the target nucleic acid is further probed with a single probe that targets a DNA fragment junction boundary.

28. The partner DNA fragment and the target DNA fragment are, respectively, AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and 20. The method of claim 19, comprising different sequences of the same gene selected from the group consisting of VEGF.

29. 20. The method of claim 19, wherein the alternative splicing event is a BCR-ABL mutation.

30. 20. The method of claim 19, wherein the third DNA fragment comprises a sequence of a partner gene or a target gene.

31. 20. The method of claim 19, wherein the signal is selected from the group consisting of a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, aminohexyl, and pyrene.

32. (a) a set of gene-specific oligonucleotides for enriching DNA to obtain a target nucleic acid, wherein the target nucleic acid is a DNA fragment linkage sequence comprising a partner DNA fragment and a target DNA fragment; (b) a split probe for probing the target nucleic acid to form separate probe-target hybrids, wherein the split probe comprises: (i) a first split probe complementary to the 3' end of the partner DNA fragment, a second split probe complementary to the 5' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp; or (ii) a first split probe complementary to the 5' end of the partner DNA fragment, a second split probe complementary to the 3' end of the target DNA fragment, and optionally a third split probe complementary to a third DNA fragment on the target nucleic acid, wherein the gap between the target site of the first split probe and the target site of the second split probe on the target nucleic acid is in the range of 0 to 80 bp. wherein the first split probe, the second split probe, and optionally the third split probe are separately immobilized to a solid support at different locations; and (c) a detectable molecule for detecting a signal at the location of the individual probe-target hybrids to identify each split probe specific to the partner DNA fragment or the target DNA fragment, wherein: (i) the partner DNA fragment is an upstream DNA fragment and the target DNA fragment is a downstream DNA fragment, or (ii) the partner DNA fragment is a downstream DNA fragment and the target DNA fragment is an upstream DNA fragment; The detectable molecule comprises a dye, a chemiluminescent dye, a fluorescent molecule, a radioisotope, a spin label, an enzyme, a hapten, a quantum dot, a bead, an aminohexyl, or a pyrene.

1. A kit for detecting a sample having a DNA fragment ligation event and / or an alternative splicing event, comprising:

33. 33. The kit of claim 32, wherein the set of gene-specific oligonucleotides is a gene-specific primer or a gene-specific probe.

34. 33. The kit of claim 32, comprising at least two pairs of gene-specific primers.

35. 33. The kit of claim 32, further comprising a universal primer.

36. 35. The kit of claim 34, wherein at least one of the gene-specific primers targets a DNA fragment junction boundary.

37. 35. The kit of claim 34, wherein the gene-specific primers target within a distance of 0 to 80 bp from a DNA fragment junction boundary.

38. 33. The kit of claim 32, wherein the first split probe or the second split probe targets within a distance of 0 to 40 bp from a DNA fragment junction boundary.

39. 33. The kit of claim 32, wherein the first split probe is selected from the group consisting of SEQ ID NOs: 32, 35, and any complementary sequence thereof.

40. 33. The kit of claim 32, wherein the second split probe is selected from the group consisting of SEQ ID NOs: 33, 36, and any complementary sequence thereof.

41. 33. The kit of claim 32, wherein the third split probe is selected from the group consisting of SEQ ID NOs: 32, 33, 35, 36, and any complementary sequence thereof.

42. 33. The kit of claim 32, wherein the split probes have a length of 10 to 60 bp.

43. 33. The kit of claim 32, further comprising a single probe that targets a DNA fragment junction boundary.

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45. 33. The kit of claim 32, wherein the second split probe is complementary to the target DNA fragment comprising a sequence of a target gene selected from the group consisting of ABL, AKT3, ALK, AXL, BCR, BRAF, CD74, ERBB2, ERBB4, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EZR, FGFR1, FGFR2, FGFR3, KIT, KMT2A, MET, NRG1, NRG2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFRA, PDGFRB, PIK3CA, RAF1, RARA, RET, ROS1, RSPO2, SDC4, SLC34A2, and TMPRSS2.

46. 33. The kit of claim 32, wherein the first split probe and the second split probe are complementary to the partner DNA fragment and the target DNA fragment, respectively, comprising different sequences of the same gene selected from the group consisting of AR, BCL2L1, BCL2L11, BCOR, BIN1, BRAF, BRCA1, BRCA2, CASP2, CD19, CD44, CXCR3, CCND1, DMP1, CDH1, EGFR, ER, EZH2, FAS, FGFR2, HRAS, IKZF1, KLF6, KRAS, MAP3K7, MCL1, MDM4, MET, MNK2, PIK3CD, PKM, RASGRP2, RON, RPS6KB, STAT3, TP53, TSC2, and VEGF.

47. 33. The kit of claim 32, wherein the DNA fragment ligation event or the alternative splicing event is a BCR-ABL mutation.

48. 33. The kit of claim 32, wherein the third split probe is complementary to the third DNA fragment comprising a sequence of a partner gene or a target gene.

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