WO2020096247A1 - Procédé de préparation d'une sonde permettant de détecter une mutation dérivée de cellules dans des tissus d'un cancer du sein et procédé de détection - Google Patents

Procédé de préparation d'une sonde permettant de détecter une mutation dérivée de cellules dans des tissus d'un cancer du sein et procédé de détection Download PDF

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WO2020096247A1
WO2020096247A1 PCT/KR2019/014241 KR2019014241W WO2020096247A1 WO 2020096247 A1 WO2020096247 A1 WO 2020096247A1 KR 2019014241 W KR2019014241 W KR 2019014241W WO 2020096247 A1 WO2020096247 A1 WO 2020096247A1
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nucleic acid
probe
sequence
gene
breast cancer
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Korean (ko)
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김효기
정재환
장훈
한원식
이한별
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주식회사 셀레믹스
서울대학교병원
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Publication of WO2020096247A1 publication Critical patent/WO2020096247A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a probe for detecting a gene mutation that can cause breast cancer, a method for manufacturing the same, and a method for detecting a gene mutation using the probe.
  • the cancer may recur in the remaining surrounding tissues and spread systemically along the blood vessels or lymphatic vessels, causing distant metastasis to other parts of the bone, lungs, liver, and brain. Therefore, additional treatment and regular check-ups are necessary to reduce recurrence.
  • somatic mutation test is required for analyzing the cause of tumor occurrence and personalized treatment.
  • the conventional method used to obtain the sample required for the test has side effects because it is an invasive method through biopsy or surgery.
  • examination costs are high and recovery time after tissue collection is long, and hospitalization is often required, and in some cases, biopsy may not be possible depending on the site of cancer.
  • a liquid biopsy such as blood or urine is non-invasive, low cost, does not require time to recover after the test, and can be applied regardless of where the cancer has occurred.
  • circulating tumor DNA which may be present in the blood of cancer patients, is present in a relatively small amount, so that whole genome sequencing or whole genome sequencing can be performed by collecting the tissue of the tumor itself. Analysis such as whole exome sequencing is not possible.
  • Targeted sequencing should be performed by selecting genes with a high probability of mutation in the test target, and in particular, ctDNA should be sophisticated enough to detect and analyze short fragments of less than 150 bp.
  • the most widely used tumor-related blood tests to date are tumor marker tests such as CEA, CA15-3, and CA125. However, tumor marker testing has been found to be less useful for diagnosis and follow-up in breast cancer.
  • a microarray or microchip is a microscopic accumulation of a large number of synthetic DNA fragments on a small slide chip.
  • a DNA fragment of a patient's sample to be analyzed is fluorescently labeled and reacted with a DNA chip, binding to an oligonucleotide (probe role) consisting of complementary sequences is made, and this is analyzed by scanning with laser light.
  • Microarrays are used in gene mutation, genotyping, and genomic analysis, and are commonly used to view single nucleotide polymorphism (SNP), indel (insertion or deletion), and rearrangement.
  • PCR polymerase chain reaction
  • This is a method of detecting a variation by amplifying a target gene region to be reported through PCR, and sequencing the resulting amplified product to analyze the base sequence.
  • This method includes the process of amplifying all regions to be viewed before sequencing, so it is possible to analyze with a small amount of DNA and has the advantage of being able to analyze the exact target gene, but making primers to amplify each region Cost, a rather high Sanger sequencing cost is required.
  • data bias may occur due to an amplification bias, which makes it difficult to detect copy number variation, and is not efficient for analyzing a wide target area.
  • the present invention is to provide a probe for detecting a gene mutation that can cause breast cancer, a method for manufacturing the same, and a method for detecting a gene mutation using the same.
  • a specific gene is selected based on the gene that causes breast cancer, and a nucleic acid (or a part thereof) constituting the gene is used as a probe, and through NGS-based target sequencing
  • a nucleic acid (or a part thereof) constituting the gene is used as a probe, and through NGS-based target sequencing
  • a probe for detecting a gene mutation capable of causing breast cancer comprising the nucleic acid of (a), (b) or (c) below or a part thereof:
  • AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA, PIK3 , RB1, TOP2A and TP53 nucleic acid comprising a base sequence constituting one or more genes selected from the group consisting of,
  • nucleic acid capable of hybridizing with a nucleic acid of (a) or a nucleic acid containing a base sequence complementary to the nucleic acid of (b) and detecting a genetic mutation that can cause breast cancer.
  • nucleic acid of (a) in the present invention AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA, PIK3R1, PTEN, RB1, TOP2A and TP53 may be nucleic acids containing nucleotide sequences constituting each gene.
  • a probe for detecting a gene mutation capable of causing breast cancer comprising the nucleic acids of (d), (e) or (f) below:
  • nucleic acid comprising the nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2285 to 4568,
  • a nucleic acid that includes a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e), and is capable of detecting gene mutations that can cause breast cancer.
  • the above-described probe is prepared, and the probe provides a method of manufacturing a probe that is sequence-designed to include a DNA sequence of an exon portion encoded by a protein. do.
  • the sequence design of the probe is designed to include all bases of the target region and a portion outside the target region, but includes a design by a 2X tiling method and an additional design of a terminal probe starting at both ends of the target. Can be.
  • the sequence may be adjusted through a GC fix process.
  • the hybridization rate of the probe may be measured to further include a re-balancing step by inserting a corresponding probe in a portion where hybridization is lower than the average hybridization rate.
  • the hybridization may further include a re-balancing step by additionally inserting the remaining probes except the corresponding probe in a portion where hybridization is higher than the average hybridization rate.
  • kits for detecting a gene mutation that can cause breast cancer including the above-described probe.
  • identification of the sequence of the sample nucleic acid may be analyzed by super-parallel sequencing.
  • the super-parallel sequencing may be any one or more selected from the group consisting of synthetic sequencing, ion torrent sequencing, pyro sequencing, sequencing by ligation, nanopore sequencing, and single-molecule real-time sequencing. have.
  • a wider target sequence of a large number of genes is accurately and at a relatively low cost. It is sensitive and can be analyzed with high reproducibility.
  • the target genes are first amplified individually and then sequenced, respectively, and thus, in the case of gene sequencing for a wide target region, efficiency is low in terms of cost and productivity.
  • the probe of the present invention instead of individual amplification of target genes, it is possible to simultaneously and repeatedly capture and select a portion to be sequenced with a small amount of DNA regardless of the length of the target region. .
  • the NGS applied in the present invention can be confirmed with high reliability for all bases of a gene, and since the base sequences are uniformly identified for all regions of the target gene, data can be accurately analyzed.
  • the configuration of the capture probe was adjusted by performing a rebalancing process for genetic regions that were not well separated.
  • the panel that has been optimized through rebalancing can provide uniform data including all target regions with a relatively small amount of sequencing data when compared to the panel without rebalancing. This can be linked to the advantage of being able to analyze accurate data at reduced cost.
  • FIG. 1 shows a schematic view of the probe design of the present invention.
  • the term 'gene' used in the present invention means a structural unit that determines genetic information, unless otherwise specified, and has a structure that has information for determining the amino acid sequence of a protein or nucleotide sequence of a functional RNA (tRNA, rRNA, etc.). Genes, and / or regulatory genes that control the expression of structural genes (eg, promoters, repressors, operators, etc.).
  • the term 'gene' as used herein is understood to mean a single-stranded side containing a nucleic acid sequence that is transcribed to produce a gene product, unless otherwise specified.
  • target gene used in the present invention may be a gene related to breast cancer, unless otherwise specified.
  • Genetic mutations that can induce breast cancer in the present invention are AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC , NF1, PIK3CA, PIK3R1, PTEN, RB1, TOP2A and TP53.
  • HGNC HUGO Gene Nomenclature Committee
  • gene mutations that may cause breast cancer may include substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to standard genomic DNA, as described above.
  • composition of the present invention may include a probe set having each sequence of SEQ ID NOs: 1 to 2284. Specifically, in the present invention, it may be used as a panel including a probe set having each sequence of SEQ ID NOs: 1 to 2284.
  • the probe set is 120 bp in length, and is constructed to overlap 0 bp to 119 bp of bases between nucleic acids constituting two probes having adjacent sequence numbers (for example, 3 of SEQ ID NO: 1 in the case of 60 bp)
  • the '60 bases at the end and the 60 bases at the 5 'end of SEQ ID NO: 2 are identical to each other, and when 0 bp are passed to the next region, even if they are adjacent sequence numbers, they do not overlap).
  • the probe set is designed to cover the CDS (coding DNA sequence) of the target gene.
  • the probe may be prepared by adjusting the sequence of the nucleic acid constituting the probe when the GC ratio is high in the target region or a specific sequence repeatedly appears to increase capture efficiency.
  • the probe set manufactured as described above is subjected to a preliminary experiment to confirm the capture efficiency, and if the capture efficiency is lower than the desired level, the number of teeth or the degree of tiling of the target area thereof is increased, and the capture efficiency is the desired level. In the higher case, it may be manufactured through a re-adjustment step of reducing the number of teeth or the degree of tiling of the target area.
  • the nucleic acid constituting the probe may be specifically DNA or RNA, and more specifically, RNA.
  • the nucleic acid of the probe that specifically hybridizes to the target gene of the present invention may be for detecting a variation in the target gene sequence. This is because these mutations can cause breast cancer.
  • the 'variation' may have a variation with respect to standard genomic DNA.
  • the variation may include a variation in the copy number of the gene or a variation in the nucleotide sequence relative to the standard genomic DNA.
  • the copy number variation of the gene may be, for example, a copy number variation (CNV).
  • Variations in the nucleotide sequence may include substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to standard genomic DNA. Substitution of the one or more nucleotide sequences may be, for example, a single nucleotide variation (SNV).
  • SNV Single Nucleotide Variation
  • Indel 'insertion-deletion mutation
  • CNV Copy Number Variation
  • 'translocation' refers to a phenomenon in which a part of a chromosome is cut, and the fragment changes to the chromosome by binding to another part of the same chromosome or another chromosome.
  • probe used in the present invention generally refers to a nucleic acid (mRNA) of a target gene in a sample that is captured by hybridization and used to detect the target nucleic acid.
  • the probe is usually a nucleic acid probe.
  • the nucleic acid constituting the probe may generally use DNA, RNA, PNA, and the like, but is not particularly limited, but DNA is preferred.
  • probe used in the present invention means a substance that specifically detects a specific substance, site, condition, etc., unless specified otherwise.
  • the nucleic acids constituting the probe include DNA, RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), zip nucleic acid (ZNA), and bridged nucleic acid (BNA) And analogs.
  • the nucleic acid constituting the probe may be specifically DNA or RNA, and more specifically, RNA.
  • the nucleic acid constituting the probe is RNA, hybridization time is shortened because the binding strength with the sample nucleic acid is superior to other strengths, and has a high detection sensitivity effect.
  • the nucleic acid constituting the probe may be synthesized using any method known in the art, for example, an automatic DNA synthesizer (eg, commercially available from BioSearch, Applied Biosystems TM, etc.).
  • the nucleic acid constituting the probe may be transcribed to generate a substance that specifically hybridizes to the mRNA of the target gene or its cDNA.
  • the transcription may be an in vitro transcription.
  • it may further include a moiety for separation or purification thereof.
  • the moiety may include one or more selected from the group consisting of biotin, avidin, and streptavidin.
  • the moiety for example, biotin, avidin, or streptavidin may include a magnetic bead, or a substance specifically binding to the moiety may include a magnetic bead.
  • the separation or purification may be achieved by a substance or magnetic field that specifically binds to the moiety.
  • a probe (or set of probes) for detecting a gene mutation that can cause breast cancer may include a nucleic acid of (a), (b) or (c) below or a part thereof:
  • AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA, PIK3 , RB1, TOP2A and TP53 nucleic acid comprising a base sequence constituting one or more genes selected from the group consisting of,
  • nucleic acid capable of hybridizing with a nucleic acid of (a) or a nucleic acid containing a base sequence complementary to the nucleic acid of (b) and detecting a genetic mutation that can cause breast cancer.
  • the nucleic acid of (a) is AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIKCA , PIK3R1, PTEN, RB1, TOP2A, and nucleic acid containing a nucleotide sequence of one or more genes selected from TP53, but may be preferably used in the following form, for example.
  • the nucleic acid of (a) is AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC , NF1, PIK3CA, PIK3R1, PTEN, RB1, TOP2A and TP53 may be nucleic acids comprising nucleotide sequences constituting each gene.
  • gene mutations that may cause breast cancer may include substitution, insertion, deletion, or translocation of one or more nucleic acid sequences relative to standard genomic DNA, as described above.
  • nucleic acid of (b) can be used for detecting gene mutations that can cause cancer in the present invention, like the nucleic acid of (a).
  • nucleic acid-related description of (a) described above can be applied in the same manner, except that the complementary base sequence of the nucleic acid of (a) is included.
  • nucleic acid of (c) can be used for detecting gene mutations that can cause breast cancer in the present invention, like the nucleic acids of (a) and (b).
  • Hybridization in the nucleic acid of (c) is, for example, a colony hybridization method or plaque hybridization as a probe for all or part of a nucleic acid containing a nucleotide sequence complementary to the base sequence of the nucleic acid of (a) or (b). Refers to what was performed using a speech method or a Southern hybridization method.
  • the hybridization can be performed at an appropriate temperature.
  • the temperature suitable for hybridization may be, for example, 40 to 80 ° C, 50 to 75 ° C, 60 to 70 ° C, or 62 to 67 ° C, and specifically 65 ° C. These hybridization temperatures are not limited thereto, and may be appropriately selected depending on the sequence and length of the polynucleotide included in the composition.
  • the hybridization time can be, for example, from 1 hour to 24 hours (overnight).
  • hybridizable nucleic acids are 70% or more, 75% or more, 80% or more, and 85% or more of the base sequence of the nucleic acid of (a) when calculated using homology search software such as BLAST. , 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, and a nucleic acid containing a nucleotide sequence having up to 99% or more homology.
  • the homology of the base sequence can be determined using the algorithm BLAST.
  • the description of the gene mutation that can cause breast cancer in the nucleic acid of (c) is described in the gene (AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1 in (a) above , FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA, PIK3R1, PTEN, RB1, TOP2A and TP53), or any of their complementary sequences It means that it can be captured by.
  • the nucleic acids of (a), (b) and (c) are not limited to the total length of the nucleic acids, and a part of them may be used as a probe.
  • Part of the nucleic acid includes, for example, a nucleic acid containing 30 to 5000 bases, a nucleic acid containing 40 to 1000 bases, a nucleic acid containing 50 to 500 bases, and a nucleic acid containing 60 to 200 bases, etc.
  • the length of the base is not particularly limited.
  • a probe for detecting a gene mutation that can cause breast cancer
  • those containing the following nucleic acids of (d), (e) or (f) may be mentioned.
  • nucleic acid comprising the nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2285 to 4568,
  • a nucleic acid that includes a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e), and is capable of detecting gene mutations that can cause breast cancer.
  • nucleic acid of (d), (e), or (f) include addition, deletion, or substituted base sequences, and also genetic mutations that can cause cancer. It may include a nucleic acid capable of detecting (hereinafter referred to as (g) nucleic acid).
  • the nucleic acid of (d) is a nucleic acid having the above-described nucleotide sequence, and the nucleotide sequence shown in the sequence number is a part of the nucleotide sequence of the gene for detecting a gene mutation that can cause cancer in the nucleic acid of (a). It is a base sequence corresponding to.
  • nucleic acid of said (d) is also preferably mentioned.
  • nucleic acid containing the nucleotide sequence as the control can also be used together with the nucleic acid of (d), and similarly, the nucleic acid of (e), (f), and (g), which will be described later, can also be used. .
  • the nucleic acid of (e), like the nucleic acid of (d), can be used for detecting gene mutations that can cause breast cancer in the present invention.
  • the nucleotide sequence shown in the sequence number is a nucleotide sequence corresponding to a part of the nucleotide sequence of the gene for detecting a gene mutation that can cause breast cancer in the nucleic acid of (a).
  • the description of the nucleic acid of (d) described above can be applied in the same manner, except that the complementary base sequence of the nucleic acid of (d) is included.
  • nucleic acid of (f) can be used for detecting gene mutations that can cause breast cancer in the present invention, like the nucleic acids of (d) and (e).
  • the nucleic acid of (f) is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more with respect to the base sequence of the nucleic acid of (d) or (e) , It is preferably a nucleic acid comprising a nucleotide sequence having a homology of 98% or more, 99% or more, and up to 99% or more.
  • the gene mutation detection capable of causing cancer is described in (a) related genes (AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1) , FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA, PIK3R1, PTEN, RB1, TOP2A and TP53), or any one of their complementary sequences by hybridization. It means you can do it.
  • nucleic acids of (g) can be used for detecting gene mutations that can cause cancer in the present invention, like the nucleic acids of (d), (e) and (f).
  • the nucleic acid of (g) is 1 to several (for example, 1 to 15, 1 to 10, or 1 to 5) in the base sequence of the nucleic acids of (d), (e) and (f), Or 1 to 2) bases are attached, deleted or substituted base sequences.
  • a base (poly T, etc.) to be a linker is modified at the end of the nucleotide sequence of the nucleic acids of (d), (e) and (f), or another base is inserted into a part of the nucleotide sequence, or And some bases of the base sequence are deleted, or some bases of the base sequence are replaced with other bases.
  • nucleic acids of (d), (e), (f) and (g) are, like the nucleic acids of (a), (b) and (c) described above, not limited to the total length of the nucleic acid, and a part thereof It can be used as a probe.
  • nucleic acids that can be used as the probe may be suitably modified.
  • a terminal vinylation (acryloylation, methacryloylation) or terminal amination is made, or a base (poly T or the like) modified as a linker.
  • other bases may be inserted into a part of the base sequence of various nucleic acids, some bases of the corresponding base sequence may be deleted, or may be replaced with a separate base or may be substituted with a substance other than the base.
  • substances other than the base include dyes (fluorescent pigments, intercalators), matting agents, and base crosslinking agents.
  • the above-described probe is prepared, and the probe provides a method of manufacturing a probe that is sequence-designed to include a DNA sequence of an exon portion encoded by a protein. do.
  • the sequence design of the probe is designed to include all bases of the target region and a portion outside the target region, but includes a design by a 2X tiling method and an additional design of a terminal probe starting at both ends of the target and on-target ratio (on The target ratio) and uniformity can be improved.
  • the tiling technique refers to sequentially designing a probe so that two or more probes with respect to a target region are overlapped with ones of the following sequence according to the length of the probe so as to target two or more probes (see FIG. 1).
  • each of the sequences of the probes constituting the set includes a sequence complementary to a partial sequence of a gene capable of detecting a gene mutation that may cause breast cancer, and a portion not targeted by the probe may not exist.
  • the entire nucleic acid sequence of the gene can be covered by the probe (s) constituting the set.
  • the term cover by the probe (s) means that the probe includes a sequence complementary to the nucleic acid sequence of the gene.
  • one or more nucleic acids of a nucleic acid sequence of a gene for detecting a gene mutation capable of causing cancer may be covered by two or more probes.
  • any probe constituting the probe set and the other probes closest thereto in order for example, 50 to 150, 60 to 140, 70 to 120, 70 to 110, 70 to 100, It may have 70 to 90, 70 to 80 identical sequences.
  • n probes when one nucleic acid of a nucleic acid sequence of a gene that detects a gene mutation capable of causing cancer is covered by n probes, it can be said to have been produced by an n x tiling technique.
  • the term 'tiling depth' may be referred to as the number of probe types that cover any target region of a gene that detects a gene mutation that can cause cancer produced by the tiling technique. It can be said that the degree of tiling increases as the species of the probe covering the target region of the gene detecting the gene mutation capable of causing cancer increases, and vice versa.
  • the sequence may be adjusted through a GC fix process.
  • GC fix process refers to adjusting the sequence of the probe by substituting the fourth base with A or T when four or more G or C are consecutive unless otherwise specified.
  • the GC fix process is a kind of a method of adjusting the polynucleic acid sequence to compensate for this, since the capture efficiency may be lowered when a polynucleotide has a high GC ratio at a position where the target gene binds or a specific sequence repeatedly appears. It is possible to provide an effect of capturing.
  • the hybridization rate of the probe is measured to further include a re-balancing step by inserting a corresponding probe in a portion where hybridization is lower than the average hybridization rate.
  • the contrast with the average hybridization rate is performed through a pilot test in which a test operation is performed in a small scale before realization in a real situation, and can be performed according to a method of analyzing the capture efficiency of a probe known in the art.
  • the hybridization may be performed by contacting and hybridizing the prepared probe set with a gene that detects a gene mutation that can cause cancer, and then sequencing the hybridization product to confirm the capture efficiency of each probe.
  • the 'capture efficiency' may be relative because the target gene sequence, for example, when the GC ratio is high in the target region or when a specific sequence repeatedly appears, or may vary depending on various factors such as a secondary structure. have. Those skilled in the art can specify the desired level of capture efficiency by referring to the above factors. Preferably, the capture efficiency is an average value of the probe sets.
  • the number of probes may be increased by further inputting the corresponding probe.
  • the degree of tiling for a target region of a gene that detects a gene mutation that can cause cancer covered by the probe may be increased.
  • the probe is manufactured by a 2x tiling technique, it may be manufactured by a 3x tiling technique, or if it is not produced by a tiling technique, a tiling technique may be introduced.
  • the number of probes can be reduced.
  • the degree of tiling of a target region of a gene that detects a gene mutation capable of causing cancer covered by the probe may be reduced. Reducing the degree of tiling may be, for example, if the corresponding probe is manufactured using a 3x tiling technique, or may be produced using a 2x tiling technique or not introduce a tiling technique.
  • the probes constituting the set of the present invention can all be included in the set in different numbers, not the same number. Likewise, the probes can all be made in different tiling techniques, not the same tiling technique, and included in a set.
  • composition of the probe set can be readjusted two or more times until the capture efficiency reaches the desired level. Through this re-adjustment step, coverage for the target area, data uniformity, and data skew can be improved.
  • kits for detecting a gene mutation that can cause breast cancer including the above-described probe.
  • the kit may further include a known substance required for the probe to hybridize with the nucleic acid of the sample.
  • reagents, buffers, cofactors, and / or substrates necessary for hybridization of nucleic acids in a sample may be further included.
  • reagents required for PCR amplification such as buffers, DNA polymerase, DNA polymerase cofactors and dNTPs may be included, and the kit also amplifies the target nucleic acid.
  • it may further include instructions for use, and may be manufactured in a number of separate packaging or compartments containing the above-described reagent components.
  • the probe may be used as a panel including a probe set.
  • the probe set is 120 bp in length, and is constructed such that 0 to 119 bp of bases overlap between nucleic acids constituting two probes having adjacent sequence numbers (for example, in the case of 60 bp, the 3 'end of SEQ ID NO: 1) 60 bases and 60 bases at the 5 'end of SEQ ID NO: 2 are identical to each other, and in the case of 0 bp, even if the sequence moves to the next region, the adjacent sequence numbers do not overlap).
  • the probe set is designed to cover a coding DNA sequence (CDS) of a gene that detects a gene mutation that may cause the cancer.
  • CDS coding DNA sequence
  • the probe (or probe set) may be prepared by adjusting the sequence of the nucleic acid constituting the probe when the GC ratio is high in a target region or a specific sequence is repeatedly displayed to increase capture efficiency.
  • the probe set manufactured as described above is subjected to a preliminary experiment to confirm the capture efficiency, and if the capture efficiency is lower than the desired level, the number of teeth or the degree of tiling of the target area thereof is increased, and the capture efficiency is the desired level. In the higher case, it may be manufactured through a re-adjustment step, which reduces the number of teeth or the degree of tiling of the target area.
  • the nucleic acid constituting the probe may be specifically DNA or RNA, and more specifically, RNA.
  • contacting the probe and the sample nucleic acid prepared in the manner described above to obtain a hybridization product of the sample nucleic acid and the probe; Identifying the sequence of the sample nucleic acid in the hybridization product; And comparing the identified nucleic acid sequence of the sample nucleic acid with a standard nucleic acid sequence to identify a variation of the sample nucleic acid.
  • the probe of the present invention can specifically hybridize to some sequences of the target gene. Some sequences of the probe may be referred to as a 'target region'.
  • the target region can be, for example, an exon or part of an exon.
  • the probe of the present invention is manufactured to have a sequence complementary to a target region, and may be annealed or hybridized with the target region under hybridization, annealing or amplification conditions.
  • 'Hybridization' of the present invention means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization can occur when the complementarity between two nucleic acid strands is a perfect match or even if some mismatch bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and may be controlled in particular by temperature.
  • the sample nucleic acid may be DNA or RNA isolated from a biological sample.
  • the biological sample may be any one or more selected from the group consisting of blood, saliva, urine, feces, tissue, cells, and biopsies.
  • the sample may be a stored biological sample or a nucleic acid isolated therefrom.
  • the storage may be stored by a known method.
  • the nucleic acid may be derived from tissue stored at room temperature in frozen storage or formalin-fixed paraffin-embedded tissue. Methods for isolating nucleic acids from biological samples are well known.
  • the sample may be isolated from the patient's cells, tissues, organs, body fluids, in which case the sample is obtained by conventional methods, for example, biopsy using methods well known by those skilled in the relevant medical technique. Can be.
  • the hybridization can be performed by a known method. For example, it can be performed by incubating the polynucleotide with a sample nucleic acid in a buffer known to be suitable for hybridization of the nucleic acid. Hybridization can be performed at an appropriate temperature.
  • the temperature suitable for hybridization may be, for example, 40 to 80 ° C, 50 to 75 ° C, 60 to 70 ° C, or 62 to 67 ° C, and specifically 65 ° C.
  • the hybridization temperature is not limited thereto, and may be appropriately selected depending on the sequence and length of the polynucleotide included in the composition.
  • the hybridization time can be, for example, from 1 hour to 24 hours (overnight).
  • next generation sequencing is a method of rapidly decoding vast amounts of genomic information by decomposing a number of fragments of the full-length genome and reading each fragment in super-parallel and then combining them using computational techniques.
  • the next-generation sequencing method can generate a large amount of sequencing data for a sample to be analyzed in a short time.
  • the super-parallel sequencing includes methods currently known as next-generation sequencing and methods that can be developed in the future.
  • the super-parallel sequencing is from a group consisting of sequencing by synthesis, ion-torrent sequencing, pyrosequencing, ligation sequencing, nanopore sequencing, and single-molecule real-time sequencing. It may be any one or more selected.
  • the probe has a nucleic acid sequence that can specifically hybridize with the target region, for example, 75 to 200, 80 to 200, 90 to 200, 100 to 200, 100 to 180, 100 to 160 Dog, 100 to 140, 100 to 120 nucleic acids in size. If the size is 75 or less, the capture accuracy of the target area is low, and when the size is 200 or more, the synthesis cost increases.
  • the probe set of the present invention can be used as a probe in target capture for targeted sequencing.
  • 'Target sequencing' refers to capturing and analyzing only the targeted region of the genome, not the entire genomic DNA, and is a representative method for confirming the variation of various genes.
  • the term 'target capture' is a method for separating and / or increasing the frequency of a particular gene or other region of interest from a DNA library prior to sequencing, where the region of interest is maintained for sequencing and the remaining material is removed.
  • a basic material called a probe is required.
  • complementary binding force between them is used.
  • RNA oligos for efficient target capture Probes synthesized in the form of nucleotides are made of RNA oligonucleotides (target capture RNA probe) through in vitro transcription. Thereafter, through the hybridization process, only specific target regions on a desired genome are captured using a probe, and the captured regions can be identified for genetic variation using next-generation sequencing technology (NGS). .
  • NGS next-generation sequencing technology
  • the detection method includes comparing the base sequence of the identified sample nucleic acid with a standard base sequence.
  • the term reference nucleic acid sequence may refer to a human gene sequence that does not contain a variation, which is referred to for identification.
  • a standard sequence a human gene sequence published in a database of the National Institute of Health Biotechnology Information (NCBI), specifically, NCBI37.1 or UCSC hg19 (GRCh37) can be used.
  • NCBI National Institute of Health Biotechnology Information
  • NCBI37.1 or UCSC hg19 GRCh37
  • the comparison between the base sequence and the standard base sequence of the sample nucleic acid can be performed using various known sequence comparison analysis programs, for example, Maq, Bowtie, SOAP, GSNAP, and the like.
  • the detection method includes the step of confirming the variation of the sample nucleic acid.
  • the mutation check may be performed using a known mutation detection program, for example, GATK, SAMtool, MoDIL, SeqSeq, PeMer, VariationHunter, Pindel, BreakDancer and Mutek, but is not limited thereto.
  • Example 1 Selection of genes related to breast cancer
  • Genes related to breast cancer, AKT1, APC, AR, BRCA1, BRCA2, CCND1, CDH1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, GATA3, IGF1R, KIT, KRAS, MAP2K4, MAP3K1, MDM2, MYC, NF1, PIK3CA PIK3R1, PTEN, RB1, TOP2A and TP53 were selected.
  • the probe was designed to include the DNA sequence for the coding sequence, that is, a portion composed of exons encoded as proteins by targeting the previously selected genes.
  • the probe was configured to include all transcripts of all genes using the UCSC genome browser.
  • a certain region (30-60 bp) was extended to the upstream on the reference genome to set the probe starting point in the intergenic region. From this, the probe was sequentially designed so that a certain portion overlaps with the next sequence according to the length of the probe. At this time, by designing with 2x tiling technique that overlaps about 60bp between neighboring probes, it is efficiently captured for all regions. To improve the on-target ratio and uniformity of the probe, a terminal probe starting at both ends was additionally designed. How to adjust the probe sequence to compensate for this, since the capture efficiency may be lowered when the probe has a high GC ratio at the position where it binds to the target gene or a specific sequence repeatedly appears. GC fix process to replace the first base with A or T) was applied to ensure efficient capture.
  • FIG. 1 shows a schematic diagram of a probe design.
  • the poorly captured portion is replenished with the probe, and the heavily captured portion is adjusted by reducing the number of probes.
  • the rebalancing was conducted. For regions with relatively low normalized depth, the same probe can be additionally inserted n times according to the depth value, and the tiling depth is increased.
  • the probe was designed by moving the probe 30bp to the periphery, thereby increasing the efficiency of capture.
  • the tiling depth was lowered or the number of probes was reduced.
  • the ratio depending on the number of probes in the section with high and low normalized depth can vary from several times to several tens of times.
  • probe sequences for each gene are shown in Table 1.
  • Probe set number Genetic name
  • Probe sequence number Probe set number Genetic name
  • Probe sequence number One AKT1 1327 ⁇ 1365 15 KIT 77 ⁇ 146 2 APC 310 ⁇ 472 16 KRAS 978 ⁇ 992 3 AR 2220 ⁇ 2284 17 MAP2K4 1582 ⁇ 1613 4 BRCA1 2097 ⁇ 2219 18 MAP3K1 147 ⁇ 251 5 BRCA2 1050 ⁇ 1254 19 MDM2 993 ⁇ 1049 6 CCND1 936 ⁇ 977 20 MYC 714 ⁇ 760 7 CDH1 1482 ⁇ 1548 21 NF1 1614 ⁇ 1829 8 EGFR 516 ⁇ 619 22 PIK3CA 1 to 76 9 ERBB2 1830 ⁇ 1958 23 PIK3R1 252 ⁇ 309 10 ESR1 473 ⁇ 515 24 PTEN 791 ⁇ 830 11 FGFR1 620 ⁇ 713 25 RB1 1255 ⁇ 1326 12 FGFR2 831 ⁇ 9
  • Human reference genome sample Prepares standard DNA (Reference DNA) using 3 types of DNA, prepares the probe set designed in Example 2, and conducts 3 gene capture experiments to improve the panel's gene capture performance and stability. The confirmed results are shown in Tables 2 and 3 below.

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Abstract

La présente invention concerne une sonde permettant de détecter une variation de gène pouvant provoquer un cancer du sein, son procédé de préparation et un procédé de détection d'une variation de gène l'utilisant. La présente invention a pour effet de détecter une variation de gène qui peut induire un cancer du sein avec précision, de manière sensible et avec une reproductibilité élevée, en fournissant un procédé pour capturer efficacement une région spécifique, même dans une petite quantité d'ADN, dans le séquençage cible basé sur du séquençage à haut débit (ou NGS, de l'anglais Next Generation Sequencing) et analyser une séquence de base.
PCT/KR2019/014241 2018-11-09 2019-10-28 Procédé de préparation d'une sonde permettant de détecter une mutation dérivée de cellules dans des tissus d'un cancer du sein et procédé de détection WO2020096247A1 (fr)

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