WO2020096248A1 - Manufacturing and detection method of probe for detecting mutations in lung cancer tissue cells - Google Patents

Manufacturing and detection method of probe for detecting mutations in lung cancer tissue cells Download PDF

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WO2020096248A1
WO2020096248A1 PCT/KR2019/014243 KR2019014243W WO2020096248A1 WO 2020096248 A1 WO2020096248 A1 WO 2020096248A1 KR 2019014243 W KR2019014243 W KR 2019014243W WO 2020096248 A1 WO2020096248 A1 WO 2020096248A1
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nucleic acid
probe
sequence
gene
hybridization
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PCT/KR2019/014243
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French (fr)
Korean (ko)
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김효기
정재환
장훈
김태민
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주식회사 셀레믹스
서울대학교병원
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Publication of WO2020096248A1 publication Critical patent/WO2020096248A1/en

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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 lung 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 and inexpensive, and does not require time to recover after the test, and can be applied regardless of the area where the cancer occurs.
  • 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.
  • 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 SNP (single nucleotide polymorphism), 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 lung 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 a gene that causes lung cancer, and a nucleic acid (or a part thereof) constituting the gene is used as a probe, and NGS-based target sequencing
  • NGS-based target sequencing By providing a method for efficiently capturing a specific region even in a small amount of DNA and analyzing the sequencing, it has been confirmed that the gene mutation can be detected accurately, sensitively and with high reproducibility, and the present invention has been completed.
  • a probe for detecting a gene mutation capable of causing lung cancer comprising the nucleic acid of (a), (b) or (c) below or a part thereof:
  • AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1 , ROS1, SETD2, STK11, TP53 and U2AF1 nucleic acid comprising a nucleotide 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 nucleotide sequence complementary to the nucleic acid of (b) and detecting gene mutations that can cause lung cancer.
  • the nucleic acid of (a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 may be nucleic acids containing nucleotide sequences constituting each gene.
  • a probe for detecting a gene mutation capable of causing lung cancer comprising the nucleic acids of (d), (e) or (f) below:
  • nucleic acid comprising a nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2341 to 4680,
  • a nucleic acid capable of detecting a gene mutation capable of causing lung cancer comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e).
  • the above-described probe is prepared, but the probe is an intron in which a DNA sequence of an exon portion encoded by a protein and a specific fusion gene mutation occur. It provides a method of manufacturing a probe that is sequence-designed to include the DNA sequence of the part.
  • 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 capable of causing lung cancer including the above-described probe.
  • 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 lung cancer, unless otherwise specified.
  • Gene mutations that can cause lung cancer in the present invention are AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA , PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1.
  • HGNC HUGO Gene Nomenclature Committee
  • gene mutations that may cause lung 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 2340. 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 2340.
  • 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 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 lung 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 acids 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 BiosystemsTM, 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 can be achieved by a substance or magnetic field that specifically binds to the moiety.
  • the probe (or set of probes) for detecting a gene mutation capable of causing lung cancer may be one comprising nucleic acids of (a), (b) or (c) below or a part thereof:
  • AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1 , ROS1, SETD2, STK11, TP53 and U2AF1 nucleic acid comprising a nucleotide 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 nucleotide sequence complementary to the nucleic acid of (b) and detecting gene mutations that can cause lung cancer.
  • the nucleic acid of (a) is AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1 , RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 is a nucleic acid comprising a nucleotide sequence of one or more genes selected from, but may be preferably used in the following form, for example.
  • the nucleic acid of (a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA , PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 may be nucleic acids comprising nucleotide sequences constituting each gene.
  • gene mutations that may cause lung 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 lung 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 that the gene mutation that can cause lung cancer in the nucleic acid of (c) is described in (a) above (AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS , KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1), or any of their complementary sequences It means that one can be captured by hybridization.
  • 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 may cause lung cancer
  • those containing the following nucleic acids of (d), (e) or (f) may be mentioned.
  • nucleic acid comprising a nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2341 to 4680,
  • a nucleic acid capable of detecting a gene mutation capable of causing lung cancer comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e).
  • 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 (d) the form containing all the nucleic acids containing each nucleotide sequence shown in 1 to 2340, for example 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) can be used for detecting gene mutations that can cause lung cancer in the present invention, like the nucleic acid of (d).
  • the base sequence shown in the sequence number is a base sequence corresponding to a part of the base sequence of the gene for detecting a gene mutation that can cause lung 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 lung 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 the aforementioned (a) related genes (AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1) , KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1). It means what can be captured by hybridization.
  • 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, but the probe is an intron in which a DNA sequence of an exon portion encoded by a protein and a specific fusion gene mutation occur. It provides a method of manufacturing a probe that is sequence-designed to include the DNA sequence of the part.
  • 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 some sequences of genes capable of detecting a gene mutation that may cause lung 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 capable of causing lung 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 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) 60 bases at the end and 60 bases at the 5 'end of SEQ ID NO: 2 are identical to each other.
  • 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 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 lung cancer
  • Genes related to lung cancer AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 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.
  • the selected genes for the ALK, BRAF, NTRK1, NTRK2, RET, and ROS1 genes, intron positions in which the fusion gene mutation occurs in the corresponding genes were selected, and a probe capable of capturing them was constructed.
  • 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 2041 ⁇ 2044 16 NF1 2079 ⁇ 2113 2 ALK 203 ⁇ 249 17 NRAS 166-174 3 ARAF 2337 ⁇ 2340 18 NTRK1 182 ⁇ 202 4 ARID1A 91 ⁇ 165 19 NTRK2 1373 ⁇ 1800 5 BRAF 1006 ⁇ 1346 20 PIK3CA 431 ⁇ 440 6 CBL 1921 ⁇ 1929 21 PTEN 1895 ⁇ 1911 7 CDKN2A 1347 ⁇ 1372 22 RB1 1934 ⁇ 2040 8 EGFR 745 ⁇ 885 23 RET 1801 ⁇ 1894 9 ERBB2 2114 ⁇ 2246 24 RIT1 175 ⁇ 181 10 HRAS 1912 ⁇ 1920 25 ROS1 441 ⁇ 744 11 KEAP1 2291 ⁇ 2330 26 SETD2 250 ⁇ 430 12 KRAS 1930 ⁇ 1933 27 STK11 2247 ⁇ 2290 13
  • 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.

Abstract

The present invention relates to a probe for detecting gene mutations capable of causing lung cancer, a method for manufacturing same and a method for detecting gene mutations by using same. According to the present invention, a method for efficiently capturing specific regions and analyzing sequences even in a small amount of DNA is provided in NGS-based target sequencing, and thus a genetic variation capable of causing lung cancer can be detected accurately, with sensitivity, and with high reproducibility.

Description

폐암 조직 내 세포 유래 돌연변이를 검출하기 위한 프로브 제조 및 검출 방법Probe preparation and detection method for detecting cell-derived mutations in lung cancer tissue
본 발명은 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브와 이를 제조하는 방법 및 이를 이용한 유전자 변이 검출 방법에 관한 것이다.The present invention relates to a probe for detecting a gene mutation that can cause lung cancer, a method for manufacturing the same, and a method for detecting a gene mutation using the probe.
암은 수술로 완전히 종양을 제거했더라도 남아있는 주변의 조직에서 재발할 수 있으며, 혈관 또는 림프관을 따라 전신적으로 퍼져 뼈, 폐, 간, 뇌 등 다른 부위에 원격전이를 일으키기도 한다. 따라서 재발을 줄이기 위해서는 추가적인 치료와 정기적인 검진이 필요하다.Even if the tumor has been completely removed by surgery, 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.
종래의 암 진단 검사에서는 종양(tumor) 발생 원인분석 및 개인 맞춤 치료 등을 위해 체세포 돌연변이(somatic mutation) 검사를 필요로 한다. 해당 검사에 필요한 시료를 얻기 위하여 현재까지 사용된 고식적 방법은 조직검사나 수술을 통한 침습적인 방법이기 때문에 부작용이 있다. 또한, 검사 비용이 높고 조직 채취 후의 회복 시간이 길어 입원 등을 요하는 경우가 많으며, 암 발생 부위에 따라 조직검사가 불가능한 경우도 있다. 반면 혈액이나 소변 등의 액체생체검사는 비침습적이며 저비용이고, 검사 후 회복하는데 시간이 필요치 않을뿐더러 암 발생 부위와 관계없이 적용이 가능하다.In the conventional cancer diagnosis test, 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. In addition, 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. On the other hand, a liquid biopsy such as blood or urine is non-invasive and inexpensive, and does not require time to recover after the test, and can be applied regardless of the area where the cancer occurs.
한편, 암환자의 혈액에 존재할 수 있는 순환종양 DNA (circulating tumor DNA, ctDNA)는 상대적으로 소량으로 존재하기 때문에 종양 자체의 조직을 채취하여 수행할 수 있는 전체염기서열분석(whole genome sequencing) 또는 전체엑솜분석(whole exome sequencing) 등의 분석은 불가능하다. 검사 대상에서 변이가 존재할 확률이 높은 유전자를 선택하여 타겟 시퀀싱(targeted sequencing)을 수행하여야 하며, 특히 ctDNA의 경우 150bp 미만의 짧은 단편(fragment)을 감지하여 분석하는 것이 가능할 정도로 정교해야 한다.On the other hand, circulating tumor DNA (ctDNA), 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.
한편, 기존에 유전자 변이를 분석하기 위한 방법으로는 마이크로어레이(microarray)를 이용하여 유전자형을 확인하는 마이크로어레이 제노타이핑(genotyping) 방법이 많이 이용되었다. 마이크로어레이 또는 마이크로 칩(DNA chip)은 작은 슬라이드 칩 상에 수많은 합성 DNA 조각을 미세하게 집적시킨 것이다. 분석하고자 하는 환자의 검체의 DNA 조각을 형광으로 표지한 후 DNA 칩에 반응시키면 상보적인 서열로 이루어져 있는 올리고뉴클레이티드(프로브 역할)와 결합이 이루어지고, 이를 레이저 빛으로 스캔하여 분석한다. 마이크로어레이는 유전자의 돌연변이, 유전형 분석, 유전체 분석 등에서 활용되고 있으며, 보통, SNP(single nucleotide polymorphism), indel(insertion 또는 deletion), 재조합(rearrangement) 등을 보는 데 이용되고 있다. 하지만 이 방법은 미량 시료에 비특이적 혼성화가 일어나면 전체 신호(signal)에 변화를 가져오기 때문에 표적으로 하는 DNA가 아닌 다른 유사한 DNA와 혼성화가 이루어지는 교차 혼성화(cross hybridization)가 발생하였을 때 정확한 유전자 분석이 어려워진다. 또한 미세한 실험 조건이나 핸들링에 의해 야기될 수 있는 결과 차이로 인한 결과 해석의 모호함, 비교적 많은 DNA 양이 필요하다는 한계점이 존재한다.Meanwhile, as a method for analyzing genetic variation, a microarray genotyping method using a microarray to identify a genotype has been widely used. A microarray or microchip (DNA chip) is a microscopic accumulation of a large number of synthetic DNA fragments on a small slide chip. When 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 SNP (single nucleotide polymorphism), indel (insertion or deletion), and rearrangement. However, since this method changes the entire signal when non-specific hybridization occurs in a trace sample, accurate genetic analysis is difficult when cross hybridization occurs that hybridizes with similar DNA other than the target DNA. Lose. In addition, there are limitations in that ambiguity in interpretation of results due to differences in results that may be caused by fine experimental conditions or handling, and relatively large amounts of DNA are required.
또한 많이 사용되고 있는 유전자 검사 방법으로는 중합효소연쇄반응(PCR)을 이용하는 방법이 있다. 이는 보고자 하는 타겟 유전자 영역에 대하여 PCR을 통해 증폭하고, 증폭된 산물을 생어 시퀀싱(Sanger sequencing)으로 염기서열을 분석하여 변이를 검출하는 방법이다. 해당 방법은 염기서열을 분석하기 전에 보고자 하는 영역을 모두 증폭하는 과정을 포함하므로 적은 DNA 양으로도 분석이 가능하며 정확한 타겟 유전자를 분석할 수 있다는 이점이 존재하지만, 각 영역을 증폭하기 위한 프라이머 제작비용, 다소 높은 생어 시퀀싱 비용이 요구된다. 또한 편향 증폭 현상 (Amplification bias)으로 인해 데이터 쏠림 현상이 발생할 수 있고 이는 단위 반복 변이(Copy number variation)를 검출하는 것이 어려워지며, 넓은 타겟 영역을 분석하기에 효율적이지 못하다.In addition, there is a method using a polymerase chain reaction (PCR) as a gene test method that is widely used. 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. In addition, 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.
선행 특허문헌Prior patent documents
KR10-2018-0042192KR10-2018-0042192
본 발명은 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브와 이를 제조하는 방법 및 이를 이용한 유전자 변이 검출 방법을 제공하고자 한다.The present invention is to provide a probe for detecting a gene mutation that can cause lung cancer, a method for manufacturing the same, and a method for detecting a gene mutation using the same.
상기 과제를 해결하기 위하여 예의 연구를 계속하던 중 폐암을 유발하는 유전자를 중심으로 특정한 유전자를 선택하고, 해당 유전자를 구성하는 핵산(또는 그의 일부)을 프로브로서 사용함과 동시에 NGS 기반의 타겟 시퀀싱을 통해 적은 양의 DNA에서도 특정 영역을 효율적으로 캡쳐하고, 염기서열을 분석하는 방법을 제공함으로써 유전자 변이를 정확하고 민감하며 높은 재현성으로 검출 가능한 것을 확인하고 본 발명을 완성하기에 이르렀다.In order to solve the above-mentioned problems, while continuing to study in earnest, a specific gene is selected based on a gene that causes lung cancer, and a nucleic acid (or a part thereof) constituting the gene is used as a probe, and NGS-based target sequencing By providing a method for efficiently capturing a specific region even in a small amount of DNA and analyzing the sequencing, it has been confirmed that the gene mutation can be detected accurately, sensitively and with high reproducibility, and the present invention has been completed.
본 발명의 일 구현예에 따르면, 하기 (a), (b) 또는 (c)의 핵산 또는 그의 일부를 포함하는 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브를 제공한다:According to one embodiment of the present invention, there is provided a probe for detecting a gene mutation capable of causing lung cancer comprising the nucleic acid of (a), (b) or (c) below or a part thereof:
(a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1으로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 구성하는 염기서열을 포함하는 핵산,(a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1 , ROS1, SETD2, STK11, TP53 and U2AF1 nucleic acid comprising a nucleotide sequence constituting one or more genes selected from the group consisting of,
(b) 상기 (a)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(b) a nucleic acid comprising a base sequence complementary to the nucleic acid of (a) above,
(c) 상기 (a)의 핵산, 또는 상기 (b)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산과 혼성화하고 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(c) A nucleic acid capable of hybridizing with a nucleic acid of (a) or a nucleic acid containing a nucleotide sequence complementary to the nucleic acid of (b) and detecting gene mutations that can cause lung cancer.
또한, 본 발명에서 상기 (a)의 핵산이 AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1의 각 유전자를 구성하는 염기서열을 포함하는 핵산일 수 있다.In addition, in the present invention, the nucleic acid of (a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 may be nucleic acids containing nucleotide sequences constituting each gene.
본 발명의 다른 구현예에 따르면, 하기 (d), (e) 또는 (f)의 핵산을 포함하는, 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브를 제공한다:According to another embodiment of the present invention, a probe for detecting a gene mutation capable of causing lung cancer is provided, comprising the nucleic acids of (d), (e) or (f) below:
(d) 서열번호 1 내지 2340의 염기서열을 포함하는 핵산,(d) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 to 2340,
(e) 서열번호 2341 내지 4680에 나타나는, 상기 (d)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(e) a nucleic acid comprising a nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2341 to 4680,
(f) 상기 (d) 또는 상기 (e)의 핵산의 염기서열에 대하여 70% 이상의 상동성을 갖는 염기서열을 포함하고, 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(f) A nucleic acid capable of detecting a gene mutation capable of causing lung cancer, comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e).
본 발명의 또 다른 구현예에 따르면, 상술한 프로브를 제조하되, 상기 프로브는 단백질로 코딩되는 엑손(exon) 부분의 DNA 시퀀스(coding sequence) 및 특정 융합(fusion) 유전자 변이가 발생하는 인트론(intron) 부분의 DNA 시퀀스를 포함하도록 시퀀스 디자인된 것인 프로브 제조방법을 제공한다.According to another embodiment of the present invention, the above-described probe is prepared, but the probe is an intron in which a DNA sequence of an exon portion encoded by a protein and a specific fusion gene mutation occur. It provides a method of manufacturing a probe that is sequence-designed to include the DNA sequence of the part.
또한, 본 발명에서 상기 프로브의 시퀀스 디자인은 타겟 영역의 모든 염기 및 타겟 영역 밖의 일부를 포함하도록 디자인되되, 2X 타일링 방법에 의한 디자인과 타겟의 양 말단에서 시작하는 터미널 프로브의 추가 디자인을 포함하는 것일 수 있다.Further, in the present invention, 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.
또한, 본 발명에서 상기 시퀀스는 GC fix 과정을 거쳐 조정된 것일 수 있다.Further, in the present invention, the sequence may be adjusted through a GC fix process.
또한, 본 발명에서 상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율 보다 낮은 부분에, 해당 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것일 수 있다.In addition, in the present invention, 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.
또한, 본 발명에서 상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율 보다 높은 부분에 해당 프로브를 제외한 나머지 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것일 수 있다.In addition, in the present invention, by measuring the hybridization rate of the probe, 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. .
본 발명의 또 다른 구현예에 있어서, 상술한 프로브를 포함하는 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 키트를 제공한다.In another embodiment of the present invention, there is provided a kit for detecting a gene mutation capable of causing lung cancer, including the above-described probe.
본 발명의 또 다른 구현예에 있어서, 상술한 프로브와 시료 핵산을 접촉시켜, 상기 시료 핵산과 상기 프로브의 혼성화 산물을 얻는 단계; 상기 혼성화 산물 중의 상기 시료 핵산의 서열을 확인하는 단계; 및 상기 시료 핵산의 확인된 핵산 서열을 표준 핵산 서열과 비교하여 상기 시료 핵산의 변이를 확인하는 단계를 포함하는 폐암을 유발할 수 있는 유전자 상의 변이를 검출하는 방법을 제공한다.In another embodiment of the present invention, contacting the above-described probe and a sample nucleic acid 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.
또한, 본 발명에서 상기 시료 핵산의 서열은 초병렬 시퀀싱에 의해 분석되는 것일 수 있다.In addition, in the present invention, the sequence of the sample nucleic acid may be analyzed by super-parallel sequencing.
또한, 본 발명에서 상기 초병렬 시퀀싱은 합성에 의한 시퀀싱, 이온 토렌트 시퀀싱, 파이로시퀀싱, 라이게이션에 의한 시퀀싱, 나노포어 시퀀싱, 및 단일-분자 실시간 시퀀싱으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것일 수 있다.In addition, in the present invention, 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.
본 발명에 따르면, 프로브와 유전자 간의 혼성화 방법을 이용한 NGS 기반의 타겟 시퀀싱 기술을 기반으로 하는 것으로, 기존의 마이크로어레이 방법이나 PCR 방법에 비해 많은 유전자의 보다 넓은 타겟 염기서열을 비교적 낮은 비용으로 정확하고 민감하며 높은 재현성으로 분석할 수 있다.According to the present invention, based on NGS-based target sequencing technology using a hybridization method between a probe and a gene, compared to a conventional microarray method or a PCR method, 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.
참고로, 기존 PCR 방법의 경우 유전적 변이를 확인하기 위해 먼저 타겟 유전자들을 개별적으로 증폭한 후 이를 각각 시퀀싱하므로 넓은 타겟 영역에 대하여 유전자 염기서열 분석을 하는 경우 비용과 생산성 면에서 효율성이 낮았다.For reference, in the case of the existing PCR method, in order to confirm the genetic variation, 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.
반면 본 발명의 프로브를 도입함으로써 타겟 유전자들의 개별적 증폭 과정 대신에 적은 양의 DNA로 염기서열 확인하고자 하는 부분을 타겟 영역의 길이에 구애받지 않고 동시 다발적으로, 정확하게 캡쳐하여 선별해내는 것이 가능하다.On the other hand, by introducing 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. .
또한, 마이크로어레이로 분석하는 경우에는 재현성 부족으로 인한 결과 해석의 모호함, 검출의 정확도가 높지 않다는 한계점이 있었다. 반면 본 발명에서 적용한 NGS는 유전자의 모든 염기에 대해 높은 신뢰성으로 확인할 수 있으며, 타겟 유전자의 모든 영역에 대하여 균일하게 염기서열을 확인하기 때문에 정확하게 데이터를 분석할 수 있다.In addition, when analyzing with a microarray, there was a limitation that the accuracy of detection and the ambiguity of interpreting the result due to insufficient reproducibility were not high. On the other hand, 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.
또한, 본 발명에 따르면, 프로브 디자인 시 타겟하는 모든 유전자에 대하여 효율적으로 캡쳐하도록 디자인 하였으나, 일부 분리가 잘 되지 않은 유전자 영역들에 대해서는 리밸런싱 과정을 진행함으로써 캡쳐 프로브의 구성을 조정하였다. 리밸런싱을 통해 최적화를 수행한 패널은 리밸런싱을 진행하지 않았을 때의 패널과 비교하였을 때, 상대적으로 적은 시퀀싱 데이터 양으로도 전 타겟 영역들을 포함하며 균일성 있는 데이터를 제공할 수 있다. 이는 절감된 비용으로 정확한 데이터를 분석할 수 있다는 이점과 연결될 수 있다.In addition, according to the present invention, when designing a probe, it was designed to efficiently capture all target genes, but 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.
도 1은 본 발명의 프로브 디자인의 모식도를 나타낸다.1 shows a schematic view of the probe design of the present invention.
이하, 본 발명에 대하여 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명에서 사용하는 용어 '유전자'는 달리 특정하지 않는 한 유전정보를 결정하는 구조 단위를 의미하는 것으로, 단백질의 아미노산 서열 또는 기능 RNA (tRNA, rRNA 등)의 염기 배열을 결정하는 정보를 가지는 구조 유전자, 및/또는 구조 유전자의 발현을 제어하는 조절 유전자 (예를 들면, 프로모터, 억제자(repressor), 작동유전자(operator) 등)를 포함한다. 본 명세서에서 사용하는 용어 '유전자'는 달리 특정하지 않는 한, 유전자의 산물을 생성하기 위해 전사되는 핵산 서열을 포함하는 단일가닥 쪽을 의미하는 것으로 이해된다.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.
본 발명에서 사용하는 용어 “표적 유전자”는 달리 특정하지 않는 한 폐암과 관련된 유전자일 수 있다.The term “target gene” used in the present invention may be a gene related to lung cancer, unless otherwise specified.
본 발명에서 폐암을 유발할 수 있는 유전자 변이는 AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1으로 이루어진 군에서 검출될 수 있다.Gene mutations that can cause lung cancer in the present invention are AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA , PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1.
상기 유전자 symbol에 해당하는 명칭 및 accession No. 에 대해서는 공개된 문헌 또는 데이터베이스를 통해 통상의 기술자에게 알려진 정보를 참조한다. 상기 공개 데이터베이스로서 HUGO Gene Nomenclature Committee(HGNC)를 참조할 수 있다.Name and accession No. corresponding to the gene symbol. For, refer to information known to those skilled in the art through published literature or databases. The HUGO Gene Nomenclature Committee (HGNC) can be referred to as the public database.
여기서, 폐암을 유발할 수 있는 유전자 변이는 전술된 바와 같이 표준 유전체 DNA에 대하여 하나 이상의 뉴클레오티드 서열의 치환, 삽입, 결실, 또는 전좌를 포함할 수 있다.Here, gene mutations that may cause lung cancer may include substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to standard genomic DNA, as described above.
본 발명의 조성물은 서열번호 1 내지 2340의 각각의 서열을 갖는 프로브 세트를 포함할 수 있다. 구체적으로, 본 발명에서는 서열번호 1 내지 2340의 각각의 서열을 갖는 프로브 세트를 포함하는 형태의 패널로 이용될 수 있다.The composition of the present invention may include a probe set having each sequence of SEQ ID NOs: 1 to 2340. 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 2340.
상기 프로브 세트는 길이가 120bp이고, 인접한 서열번호를 갖는 2개의 프로브를 구성하는 핵산들 간에 0~119bp의 염기가 겹치도록 제작된 것이다(예를 들면 60bp인 경우는 서열번호 1의 3' 말단의 60개의 염기와 서열번호 2의 5' 말단의 60개의 염기가 서로 동일하고, 0bp인 경우는 다음 영역으로 넘어가는 경우 인접 서열번호여도 겹치지 않음). 또한 상기 프로브 세트는 해당 표적 유전자의 CDS(coding DNA sequence)를 커버할 수 있도록 제작된 것이다. 또한, 상기 프로브는, 캡쳐 효율을 높이기 위해 타겟 영역에 GC 비율이 높거나 특정 서열이 반복적으로 나타날 경우, 프로브를 구성하는 핵산의 서열을 조정하여 제작된 것일 수 있다.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). In addition, the probe set is designed to cover the CDS (coding DNA sequence) of the target gene. In addition, 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.
상기된 바와 같이 제작된 프로브 세트는 캡쳐 효율을 확인하는 예비 실험을 실시하여, 캡쳐 효율이 목적한 수준보다 낮은 경우에는 이의 개수 또는 이의 타겟 영역에 대한 타일링 정도를 증가시키고, 캡쳐 효율이 목적한 수준보다 높은 경우에는 이의 개수 또는 이의 타겟 영역에 대한 타일링 정도를 감소시키는 재조정 단계를 거쳐서 제조된 것일 수 있다.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.
상기 프로브를 구성하는 핵산은 구체적으로 DNA 또는 RNA일 수 있으며, 더욱 구체적으로는 RNA일 수 있다.The nucleic acid constituting the probe may be specifically DNA or RNA, and more specifically, RNA.
본 발명의 표적 유전자에 특이적으로 혼성화하는 프로브의 핵산은 표적 유전자 서열의 변이를 검출하기 위한 것일 수 있다. 이러한 변이가 폐암을 유발할 수 있기 때문이다.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 lung cancer.
상기 '변이'는 표준 유전체 DNA에 대하여 변이를 갖는 것일 수 있다. 구체적으로 상기 변이는 표준 유전체 DNA에 대하여 유전자의 카피 수의 변이 또는 뉴클레오티드 서열의 변이를 포함할 수 있다. 상기 유전자의 카피수의 변이는 예를 들면 복제수변이(CNV)일 수 있다. 상기 뉴클레오티드 서열의 변이는 표준 유전체 DNA에 대하여 하나 이상의 뉴클레오티드 서열의 치환, 삽입, 결실, 또는 전좌를 포함할 수 있다. 상기 하나 이상의 뉴클레오티드 서열의 치환은 예를 들면 단일 뉴클레오티드 변이(SNV)일 수 있다.The 'variation' may have a variation with respect to standard genomic DNA. Specifically, 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).
용어 '단일 뉴클레오티드 변이 (Single Nucleotide Variation: SNV)'는 단일 뉴클레오티드 다형성(Single Nucleotide Polymorphism)이 하나의 종내 다수의 집단에서 나타나는 단일염기의 차이를 말하는 것에 비해, 하나의 서열 또는 종 내 소수의 집단에서 나타나는 단일염기의 차이를 의미하는 것으로, 예를 들면 시퀀싱 데이터에서 나타나는 표준염기서열과의 차이를 의미할 수 있다.The term 'Single Nucleotide Variation (SNV)' refers to the difference between a single base in a single nucleotide polymorphism and multiple populations in a single species, compared to a single nucleotide variation (single nucleotide variation). It means the difference between the single bases, and for example, the difference from the standard base sequence shown in the sequencing data.
용어 '삽입-결실 변이(Indel)'는 유전자의 핵산 개수를 변화시킬 수 있는 염기 서열이 삽입 또는 결실된 것을 의미한다.The term 'insertion-deletion mutation (Indel)' means that a base sequence capable of changing the number of nucleic acids in a gene is inserted or deleted.
용어 '복제수 변이(Copy Number Variation: CNV)'는 특정 염색체의 상대적으로 큰 영역이 결손되거나 증폭되어 반복적으로 나타나는 유전체 DNA의 변이를 의미하는 것으로, 예를 들면 1kB 이상의 DNA 조각이 중첩되어 존재하거나 일부가 결실되는 변이일 수 있다.The term 'Copy Number Variation (CNV)' refers to a mutation in a genomic DNA that repeatedly appears when a relatively large region of a specific chromosome is deleted or amplified. For example, DNA fragments of 1 kB or more are overlapped or present. It may be a variant in which some are deleted.
용어 '전좌(translocation)'는 염색체의 일부분에 절단이 일어나, 그 단편이 동일 염색체의 다른 부분 또는 다른 염색체에 결합하여 염색체의 형태를 바꾸는 현상을 의미한다.The term '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.
본 발명에서 사용하는 용어 “프로브”란 일반적으로 검체 중의 표적으로 하는 유전자의 핵산(mRNA)을 혼성화에 의해 포착하고, 당해 표적 핵산을 검출하기 위해 사용되는 것을 지칭한다. 상기 프로브는 통상 핵산 프로브인데, 본 발명에서 프로브를 구성하는 핵산은 일반적으로 DNA, RNA, PNA 등을 사용할 수 있고, 특히 한정하는 것은 아니나 DNA가 바람직하다. 본 발명에서 사용하는 용어 “프로브”는 달리 특정하지 않는 한 특정 물질, 부위, 상태 등을 특이적으로 검출하는 물질을 의미한다.The term “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. In the present invention, the nucleic acid constituting the probe may generally use DNA, RNA, PNA, and the like, but is not particularly limited, but DNA is preferred. The term “probe” used in the present invention means a substance that specifically detects a specific substance, site, condition, etc., unless specified otherwise.
상기 프로브를 구성하는 핵산은 DNA, RNA, 펩티드 핵산(Peptide Nucleic Acid: PNA), 잠금 핵산(Locked Nucleic Acid: LNA), 지프 핵산(Zip Nucleic Acid: ZNA), 가교 핵산(Bridged Nucleic Acid: BNA) 및 유사체로부터 선택되는 하나 이상일 수 있다. 상기 프로브를 구성하는 핵산은 구체적으로 DNA 또는 RNA일 수 있으며, 더욱 구체적으로는 RNA일 수 있다. 상기 프로브를 구성하는 핵산이 RNA일 경우, 시료 핵산과의 결합 강도가 다른 강도보다 우수하여 혼성화 시간이 단축되고, 높은 검출 민감도의 효과를 갖는다.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. When 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.
상기 프로브를 구성하는 핵산은 본 분야에서 공지된 임의의 방법, 예를 들면 자동 DNA 합성기 (예, 바이오서치, 어플라이드 바이오시스템TM 등으로 구입할 수 있는 것)를 사용하여 합성될 수 있다. 상기 프로브를 구성하는 핵산은 전사됨으로써 표적 유전자의 mRNA 또는 그의 cDNA에 특이적으로 혼성화하는 물질을 생성하는 것일 수 있다. 상기 전사는 인 비트로 전사일 수 있다. 또한 이의 분리 또는 정제를 위한 모이어티(moiety)를 더 포함할 수 있다. 상기 모이어티는 비오틴, 아비딘, 및 스트렙타비딘으로 이루어진 군으로부터 선택되는 하나 이상을 포함할 수 있다. 또한 상기 모이어티, 예를 들면 비오틴, 아비딘 또는 스트렙타비딘은 자성비드(magnetic bead)를 포함하거나, 또는 상기 모이어티에 특이적으로 결합하는 물질이 자성비드를 포함할 수 있다. 상기 분리 또는 정제는 모이어티에 특이적으로 결합하는 물질 또는 자기장에 의해 이루어질 수 있다.The nucleic acids 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 BiosystemsTM, 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. In addition, 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. In addition, 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 can be achieved by a substance or magnetic field that specifically binds to the moiety.
본 발명에서, 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브(또는 프로브 세트)는 하기 (a), (b) 또는 (c)의 핵산 또는 그의 일부를 포함하는 것일 수 있다:In the present invention, the probe (or set of probes) for detecting a gene mutation capable of causing lung cancer may be one comprising nucleic acids of (a), (b) or (c) below or a part thereof:
(a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1으로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 구성하는 염기서열을 포함하는 핵산,(a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1 , ROS1, SETD2, STK11, TP53 and U2AF1 nucleic acid comprising a nucleotide sequence constituting one or more genes selected from the group consisting of,
(b) 상기 (a)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(b) a nucleic acid comprising a base sequence complementary to the nucleic acid of (a) above,
(c) 상기 (a)의 핵산, 또는 상기 (b)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산과 혼성화하고 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(c) A nucleic acid capable of hybridizing with a nucleic acid of (a) or a nucleic acid containing a nucleotide sequence complementary to the nucleic acid of (b) and detecting gene mutations that can cause lung cancer.
상기 (a)의 핵산이 AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1으로부터 선택되는 1종 이상의 유전자의 염기서열을 포함하는 핵산이지만, 예를 들면 이하의 형태로 사용하는 것이 바람직할 수 있다. 예를 들면, 상기 (a)의 핵산이 AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1의 각 유전자를 구성하는 염기서열을 포함하는 핵산일 수 있다. 여기서, 폐암을 유발할 수 있는 유전자 변이는 전술된 바와 같이 표준 유전체 DNA에 대하여 하나 이상의 핵산 서열의 치환, 삽입, 결실, 또는 전좌를 포함할 수 있다.The nucleic acid of (a) is AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1 , RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 is a nucleic acid comprising a nucleotide sequence of one or more genes selected from, but may be preferably used in the following form, for example. For example, the nucleic acid of (a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA , PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 may be nucleic acids comprising nucleotide sequences constituting each gene. Here, gene mutations that may cause lung cancer may include substitution, insertion, deletion, or translocation of one or more nucleic acid sequences relative to standard genomic DNA, as described above.
또한, 상기 (b)의 핵산도, 상기 (a)의 핵산과 마찬가지로, 본 발명에 있어 암을 유발할 수 있는 유전자 변이 검출 등에 사용할 수 있다. 상기 (b)의 핵산에 대해서는 상기 (a)의 핵산의 상보쇄 염기서열을 포함하는 것 이외에는 상술한 (a)의 핵산 관련 설명을 동일하게 적용할 수 있다.In addition, the 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). Regarding the nucleic acid of (b), the 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.
또한, 상기 (c)의 핵산도, 상기 (a) 및 (b)의 핵산과 마찬가지로, 본 발명에 있어 폐암을 유발할 수 있는 유전자 변이 검출 등에 사용할 수 있다. 상기 (c)의 핵산에서 혼성화는 예를 들면, 상기 (a)의 핵산 또는 (b)의 핵산의 염기서열과 상보적인 염기서열을 포함하는 핵산의 전부 또는 일부를 프로브로서 콜로니 혼성화법, 플라크 혼성화법 또는 서던 혼성화법 등을 사용하여 수행한 것을 지칭한다.In addition, the nucleic acid of (c) can be used for detecting gene mutations that can cause lung 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.
이러한 혼성화에 영향을 미치는 요소로는 온도, 프로브 농도, 프로브 길이, 반응 시간, 이온 강도, 염 농도 등 복수의 요소가 고려될 수 있다. 상기 혼성화는 적절한 온도에서 수행될 수 있다. 혼성화에 적절한 온도는 예를 들면, 40 내지 80℃, 50 내지 75℃, 60 내지 70℃, 또는 62 내지 67℃일 수 있으며, 구체적으로는 65℃일 수 있다. 이들 혼성화 온도는 이에 제한되지 않고, 조성물에 포함된 폴리뉴클레오티드의 서열 및 길이에 따라 적절하게 선택될 수 있다. 혼성화 시간은 예를 들면, 1 시간 내지 24시간 (밤새) 동안일 수 있다.As factors influencing hybridization, a plurality of factors such as temperature, probe concentration, probe length, reaction time, ionic strength, and salt concentration may be considered. 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).
이들 이외에 혼성화 가능한 핵산으로는, BLAST 등의 상동성 검색 소프트웨어에 의해 디폴트의 파라미터를 이용하여 계산 시 상기 (a)의 핵산의 염기서열과 70% 이상, 75% 이상, 80% 이상, 85% 이상, 90% 이상, 95% 이상, 97% 이상, 98% 이상, 99% 이상, 최대 99% 이상의 상동성을 갖는 염기서열을 포함하는 핵산을 들 수 있다.In addition to these, 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.
또한, 염기서열의 상동성은 알고리즘 BLAST을 사용하여 결정할 수 있다. 관련 프로그램으로 BLASTN이나 BLASTX 등이 있다. BLASTN을 사용하여 염기서열을 해석하는 경우에는 파라미터는 score=100, word length=12fh 한다. 또한 상기 (c)의 핵산에 있어서 폐암을 유발할 수 있는 유전자 변이를 검출한다는 기재는 상기 (a)에 있어 열거한 유전자(AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1)의 염기서열이나 그의 상보쇄 염기서열 중 어느 하나를 혼성화에 의해 포착할 수 있는 것을 의미한다.In addition, the homology of the base sequence can be determined using the algorithm BLAST. Related programs include BLASTN and BLASTX. When nucleotide sequences are analyzed using BLASTN, the parameters are score = 100 and word length = 12fh. In addition, the description that the gene mutation that can cause lung cancer in the nucleic acid of (c) is described in (a) above (AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS , KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1), or any of their complementary sequences It means that one can be captured by hybridization.
상기 (a), (b) 및 (c)의 핵산은 당해 핵산의 전체 길이에 한정되지 않고, 그의 일부를 프로브로서 사용할 수도 있다. 상기 핵산의 일부란, 예를 들면 30 내지 5000 염기를 포함하는 핵산, 40 내지 1000 염기를 포함하는 핵산, 50 내지 500 염기를 포함하는 핵산, 나아가 60 염기 내지 200 염기를 포함하는 핵산 등을 들 수 있으나, 염기 길이에 대해 특별히 한정되는 것은 아니다.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. However, the length of the base is not particularly limited.
또한, 본 발명에서 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브(또는 프로브 세트)로는, 예를 들면, 이하의 (d), (e) 또는 (f)의 핵산을 포함하는 것도 들 수 있다:In addition, in the present invention, as a probe (or set of probes) for detecting a gene mutation that may cause lung cancer, for example, those containing the following nucleic acids of (d), (e) or (f) may be mentioned. :
(d) 서열번호 1 내지 2340의 염기서열을 포함하는 핵산,(d) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 to 2340,
(e) 서열번호 2341 내지 4680에 나타나는, 상기 (d)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(e) a nucleic acid comprising a nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2341 to 4680,
(f) 상기 (d) 또는 상기 (e)의 핵산의 염기서열에 대하여 70% 이상의 상동성을 갖는 염기서열을 포함하고, 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(f) A nucleic acid capable of detecting a gene mutation capable of causing lung cancer, comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e).
여기에 추가로 상기 (d), (e), 또는 (f)의 핵산의 염기서열에 있어 1 내지 수개의 염기가 부가, 결실 또는 치환된 염기서열을 포함하고, 또한 암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산(이하 (g)의 핵산이라 칭함)을 포함할 수 있다.In addition, 1 to several bases in the base sequence of the 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).
상기 (d)의 핵산은, 상술한 염기서열을 갖는 핵산이고, 상기 서열번호에 나타나는 염기서열은 상기 (a)의 핵산에 있어서 암을 유발할 수 있는 유전자 변이를 검출하기 위한 유전자의 염기서열의 일부에 해당하는 염기서열이다.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.
또한, 상기 (d)의 핵산으로는, 예를 들면 1 내지 2340에 나타나는 각 염기서열을 포함하는 핵산을 모두 포함하는 형태도 바람직하게 들 수 있다.Moreover, as the nucleic acid of (d), the form containing all the nucleic acids containing each nucleotide sequence shown in 1 to 2340, for example is also preferably mentioned.
본 발명에서 상기 컨트롤로서의 염기서열을 포함하는 핵산도, 상기 (d)의 핵산과 함께 사용할 수 있고, 마찬가지로 후술하는 (e), (f)의 핵산, 나아가 (g)의 핵산과 함께 사용할 수도 있다.In the present invention, the 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. .
상기 (e)의 핵산도, 상기 (d)의 핵산과 마찬가지로, 본 발명에서 폐암을 유발할 수 있는 유전자 변이 검출 등에 사용할 수 있다. 상기 서열 번호에 나타나는 염기서열은 상기 (a)의 핵산에 있어서 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 유전자의 염기서열의 일부에 해당하는 염기서열이다. 상기 (e)의 핵산에 대해서는, 상기 (d)의 핵산의 상보쇄 염기서열을 포함하는 것 이외에는 상술한 (d)의 핵산에 관한 설명을 동일하게 적용할 수 있다.The nucleic acid of (e) can be used for detecting gene mutations that can cause lung cancer in the present invention, like the nucleic acid of (d). The base sequence shown in the sequence number is a base sequence corresponding to a part of the base sequence of the gene for detecting a gene mutation that can cause lung cancer in the nucleic acid of (a). For the nucleic acid of (e), 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.
또한, 상기 (f)의 핵산도, 상기 (d) 및 (e)의 핵산과 마찬가지로, 본 발명에서 폐암을 유발할 수 있는 유전자 변이 검출 등에 사용할 수 있다. In addition, the nucleic acid of (f) can be used for detecting gene mutations that can cause lung cancer in the present invention, like the nucleic acids of (d) and (e).
상기 (f)의 핵산은 상기 (d) 또는 (e)의 핵산의 염기서열에 대하여, 70% 이상, 75% 이상, 80% 이상, 85% 이상, 90% 이상, 95% 이상, 97% 이상, 98% 이상, 99% 이상, 최대 99% 이상의 상동성을 갖는 염기서열을 포함하는 핵산인 것이 바람직하다.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.
또한 상기 (f)의 핵산에 있어서, 암을 유발할 수 있는 유전자 변이검출이란 전술한 (a) 관련 열거한 유전자(AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1)의 염기서열이나 그의 상보쇄 염기서열 중 어느 하나를 혼성화에 의해 포착할 수 있는 것을 의미한다.In addition, in the nucleic acid of (f), the gene mutation detection capable of causing cancer is the aforementioned (a) related genes (AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1) , KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1). It means what can be captured by hybridization.
또한, 상기 (g)의 핵산도 상기 (d), (e) 및 (f)의 핵산과 마찬가지로, 본 발명에서 암을 유발할 수 있는 유전자 변이 검출 등에 사용할 수 있다.In addition, the 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).
상기 (g)의 핵산은 상기 (d), (e) 및 (f)의 핵산의 염기서열에 있어서 1 내지 수개(예를 들면, 1 내지 15개, 1 내지 10개, 또는 1 내지 5개, 또는 1 내지 2개)의 염기가 부착, 결실 또는 치환된 염기서열이다. 예를 들면, 상기 (d), (e) 및 (f)의 핵산의 염기서열 말단에 링커가 되는 염기(폴리T 등)를 개질한 것이나, 해당 염기서열의 일부에 다른 염기를 삽입하거나, 해당 염기서열의 일부 염기를 결실시키거나, 해당 염기서열의 일부 염기를 다른 염기로 치환한 것 등을 들 수 있다.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. For example, 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.
상기 (d), (e), (f) 및 (g)의 핵산은 상술한 (a), (b) 및 (c)의 핵산과 마찬가지로, 해당 핵산의 전체 길이에 한정되지 않고, 그의 일부를 프로브로서 사용할 수 있다.The 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.
상기 프로브로서 사용할 수 있는 각종 핵산은 적절하게 개질된 것일 수 있다. 일예로, 말단 비닐화(아크릴로일화, 메타크릴로일화) 또는 말단 아미노화가 이루어진 것이나, 링커가 되는 염기(폴리T 등)로 수식된 것 등을 들 수 있다. 또한, 각종 핵산의 염기서열 일부에 다른 염기를 삽입하거나, 해당 염기서열의 일부 염기를 결실시키거나, 또는 별도의 염기로 치환하거나, 또는 염기 이외의 물질로 치환하여 사용할 수 있다. 여기서 염기 이외의 물질이란 예를 들면 색소(형광색소, 인터칼레이터), 소광제, 염기 가교제 등을 들 수 있다.Various nucleic acids that can be used as the probe may be suitably modified. As an example, a terminal vinylation (acryloylation, methacryloylation) or terminal amination is made, or a base (poly T or the like) modified as a linker. Further, 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. Examples of substances other than the base include dyes (fluorescent pigments, intercalators), matting agents, and base crosslinking agents.
본 발명의 또 다른 구현예에 따르면, 상술한 프로브를 제조하되, 상기 프로브는 단백질로 코딩되는 엑손(exon) 부분의 DNA 시퀀스(coding sequence) 및 특정 융합(fusion) 유전자 변이가 발생하는 인트론(intron) 부분의 DNA 시퀀스를 포함하도록 시퀀스 디자인된 것인 프로브 제조방법을 제공한다.According to another embodiment of the present invention, the above-described probe is prepared, but the probe is an intron in which a DNA sequence of an exon portion encoded by a protein and a specific fusion gene mutation occur. It provides a method of manufacturing a probe that is sequence-designed to include the DNA sequence of the part.
상기 프로브의 시퀀스 디자인은 타겟 영역의 모든 염기 및 타겟 영역 밖의 일부를 포함하도록 디자인되되, 2X 타일링 방법에 의한 디자인과 타겟의 양 말단에서 시작하는 터미널 프로브의 추가 디자인을 포함하고 온-타겟 비(on target ratio) 및 균일성(uniformity) 향상을 도모할 수 있다.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.
상기 타일링(tiling) 기법은 타겟 영역에 대하여 2이상의 프로브가 둘 이상의 타겟하도록 프로브의 길이에 따라 일정 부분이 다음 순서의 것과 겹치도록 프로브를 순차 디자인한 것을 지칭한다(도 1 참조). 이 경우 세트를 구성하는 프로브의 서열 각각은 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 유전자의 일부 서열에 상보적인 서열을 포함하며, 프로브에 의해 타겟 되지 않는 부분은 존재하지 않을 수 있다. 이는 해당 유전자 전체 핵산 서열이 세트를 구성하는 프로브(들)에 의해 커버될 수 있음을 의미하다. 여기서 용어 프로브(들)에 의한 커버는 프로브가 유전자의 핵산 서열에 상보적인 서열을 포함하는 것을 의미한다.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). In this case, each of the sequences of the probes constituting the set includes a sequence complementary to some sequences of genes capable of detecting a gene mutation that may cause lung cancer, and a portion not targeted by the probe may not exist. This means that the entire nucleic acid sequence of the gene can be covered by the probe (s) constituting the set. Here, the term cover by the probe (s) means that the probe includes a sequence complementary to the nucleic acid sequence of the gene.
상기 타일링 기법으로 제작된 프로브 세트의 경우, 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 핵산 서열 중 한 개의 핵산이 2 종 이상, 구체적으로는 3 종 이상의 프로브에 의해 커버될 수 있다.In the case of a probe set produced by the tiling technique, 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.
또한, 이 경우, 프로브 세트를 구성하는 임의의 프로브와 순서상 이와 가장 인접한 다른 프로브는 예를 들면 50 내지 150개, 60 내지 140개, 70 내지 120개, 70 내지 110개, 70 내지 100개, 70 내지 90개, 70 내지 80개의 동일한 서열을 가질 수 있다.Also, in this case, 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.
본 발명에서 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 핵산 서열 중 한 개의 핵산이 n종의 프로브에 의해 커버될 때 n x 타일링 기법으로 제조된 것이라 할 수 있다. 용어 '타일링 정도(tiling depth)'는 상기 타일링 기법으로 제조된 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 임의의 타겟 영역을 커버하는 프로브 종류의 개수라 할 수 있다. 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 타겟 영역을 커버하는 프로브의 종이 증가할수록 타일링 정도는 높아지고, 이와 반대로 커버하는 프로브의 종이 감소할수록 타일링 정도는 낮아진다고 할 수 있다.In the present invention, 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.
상기 시퀀스는 GC fix 과정을 거쳐 조정된 것일 수 있다. 여기서 사용하는 용어 “GC fix 과정”은 달리 특정하지 않는 한 G 또는 C가 4개 이상 연속될 때에 4번째 염기를 A 또는 T로 치환하여 프로브의 시퀀스를 조정한 것을 지칭한다. 상기 GC fix 과정은 폴리뉴클레오티드가 표적 유전자와 결합하는 위치에 높은 GC ratio를 지니거나 특정 서열이 반복적으로 나타날 때 캡쳐 효율이 낮아질 수 있으므로 이를 보완하기 위하여 폴리핵산 서열을 조정하는 방식의 일종으로, 효율적인 캡쳐가 이루어지는 효과를 제공할 수 있다. The sequence may be adjusted through a GC fix process. As used herein, the term “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.
또한, 본 발명에서 상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율보다 낮은 부분에, 해당 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것이 바람직하다.In addition, in the present invention, it is preferable that 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.
또한, 본 발명에서 상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율보다 높은 부분에, 해당 프로브를 제외한 나머지 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것이 바람직하다.In addition, in the present invention, it is preferable to further include a re-balancing step by measuring the hybridization rate of the probe and inserting the remaining probes excluding the probe to the portion where hybridization is higher than the average hybridization rate. Do.
이러한 평균 혼성화율과의 대비는 실제 상황에서 실현하기 전에 소규모로 시험 작동해보는 예비 실험(pilot test)을 통해 수행되는 것으로, 당해 분야에 알려진 프로브의 캡쳐 효율을 분석하는 방법에 따라 실시할 수 있다. 예를 들면, 상기 제작된 프로브 세트와 암을 유발할 수 있는 유전자 변이를 검출하는 유전자를 접촉하여 혼성화시킨 후 이 혼성화 산물을 분리하여 시퀀싱함으로써 각 프로브의 캡쳐 효율을 확인할 수 있다.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. For example, 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.
본 발명에서 '캡쳐 효율'은 표적 유전자의 서열, 예를 들면 타겟 영역에 GC 비율이 높거나 특정 서열이 반복적으로 나타날 경우, 혹은 이차 구조(secondary structure) 등 여러 요인에 따라 달라질 수 있으므로 상대적일 수 있다. 당업자라면 상기 요인들을 참조하여 목적한 수준의 캡쳐 효율을 특정할 수 있다. 상기 캡쳐 효율은 프로브 세트들의 캡쳐 효율에 대한 평균값이 것이 바람직하다.In the present invention, 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.
상기 예비 실험을 통해 확인된 캡쳐 효율이 목적한 수준보다 낮은 경우에는 해당 프로브를 더욱 투입하여 개수를 증가시킬 수 있다. 또는 상기 프로브에 의해 커버되는 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 타겟 영역에 대한 타일링 정도를 증가시킬 수 있다. 예를 들어 해당 프로브가 2 x 타일링 기법으로 제작된 것이라면 3 x 타일링 기법으로 제작하는 것일 수 있고, 타일링 기법에 의해 제작되지 않았다면 타일링 기법을 도입하는 것일 수 있다.If the capture efficiency confirmed through the preliminary experiment is lower than the desired level, the number of probes may be increased by further inputting the corresponding probe. Alternatively, 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. For example, if 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.
반대로, 캡쳐 효율이 목적한 수준보다 높은 경우에는 해당 프로브의 개수를 감소시킬 수 있다. 또는 상기 프로브에 의해 커버되는 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 타겟 영역에 대한 타일링 정도를 감소시킬 수 있다. 타일링 정도를 감소시킨다는 것은, 예를 들어 해당 프로브가 3 x 타일링 기법으로 제작된 것이라면 2 x 타일링 기법으로 제작하거나, 타일링 기법을 도입하지 않는 것일 수 있다.Conversely, if the capture efficiency is higher than the desired level, the number of probes can be reduced. Alternatively, 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.
이러한 재조정 단계를 거침으로써, 본 발명의 세트를 구성하는 프로브들은 모두 동일한 개수가 아닌 서로 다른 개수로 세트에 포함될 수 있다. 마찬가지로 프로브들은 모두 동일한 타일링 기법이 아닌 서로 다른 타일링 기법으로 제작되어 세트에 포함될 수 있다.By going through this readjustment step, 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.
캡쳐 효율이 목적한 수준에 이를 때까지 프로브 세트의 조성을 2회 이상 재조정할 수 있다. 이러한 재조정 단계를 통해 타겟 영역에 대한 커버리지, 데이터 균일성, 데이터 쏠림 현상을 개선할 수 있다.The 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.
본 발명의 또 다른 구현예에 따르면, 상술한 프로브를 포함하는, 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 키트를 제공한다. 상기 키트는 상기 프로브가 시료의 핵산과 혼성화 하는데 요구되는 알려진 물질을 더 포함할 수 있다. 예를 들면, 시료 내 핵산의 혼성화에 필요한 시약, 버퍼, 보조인자, 및/또는 기질을 더 포함할 수 있다. 또한 상기 키트가 PCR 증폭 과정에 적용되는 경우 선택적으로, PCR 증폭에 필요한 시약, 예컨대, 완충액, DNA 중합효소, DNA 중합효소보조인자 및 dNTPs를 포함할 수 있으며, 또한, 상기 키트는 표적 핵산을 증폭하기 위하여 사용하기 위한 설명서를 더 포함할 수 있으며, 상기한 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다. 본 발명에서 상기 프로브는 프로브 세트를 포함하는 형태의 패널로 이용될 수 있다.According to another embodiment of the present invention, there is provided a kit for detecting a gene mutation capable of causing lung 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. For example, reagents, buffers, cofactors, and / or substrates necessary for hybridization of nucleic acids in a sample may be further included. In addition, when the kit is applied to a PCR amplification process, optionally, 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. In order to do so, 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. In the present invention, the probe may be used as a panel including a probe set.
상기 프로브 세트는 길이가 120bp이고, 인접한 서열번호를 갖는 2개의 프로브를 구성하는 핵산들 간에 0 bp 내지 119 bp의 염기가 겹치도록 제작된 것이다(예를 들면 60bp인 경우는 서열번호 1의 3' 말단의 60개의 염기와 서열번호 2의 5' 말단의 60개의 염기가 서로 동일하고, 0bp인 경우는 다음 영역으로 넘어가는 경우 인접 서열번호여도 겹치지 않음). 또한 상기 프로브 세트는 해당 암을 유발할 수 있는 유전자 변이를 검출하는 유전자의 CDS(coding DNA sequence)를 커버할 수 있도록 제작된 것이다.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) 60 bases at the end and 60 bases at the 5 'end of SEQ ID NO: 2 are identical to each other. In addition, 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.
또한, 상기 프로브(또는 프로브 세트)는, 캡쳐 효율을 높이기 위해 타겟 영역에 GC 비율이 높거나 특정 서열이 반복적으로 나타날 경우, 프로브를 구성하는 핵산의 서열을 조정하여 제작된 것일 수 있다.In addition, 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.
상기된 바와 같이 제작된 프로브 세트는 캡쳐 효율을 확인하는 예비 실험을 실시하여, 캡쳐 효율이 목적한 수준보다 낮은 경우에는 이의 개수 또는 이의 타겟 영역에 대한 타일링 정도를 증가시키고, 캡쳐 효율이 목적한 수준보다 높은 경우에는 이의 개수 또는 이의 타겟 영역에 대한 타일링 정도를 감소시키는, 재조정 단계를 거쳐서 제조된 것일 수 있다.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.
상기 프로브를 구성하는 핵산은 구체적으로 DNA 또는 RNA일 수 있으며, 더욱 구체적으로는 RNA일 수 있다.The nucleic acid constituting the probe may be specifically DNA or RNA, and more specifically, RNA.
본 발명의 또 다른 구현예에 있어서, 전술한 방식으로 제조된 프로브와 시료 핵산을 접촉시켜, 상기 시료 핵산과 상기 프로브의 혼성화 산물을 얻는 단계; 상기 혼성화 산물 중의 상기 시료 핵산의 서열을 확인하는 단계; 및 상기 시료 핵산의 확인된 핵산 서열을 표준 핵산 서열과 비교하여 상기 시료 핵산의 변이를 확인하는 단계를 포함하는 폐암을 유발할 수 있는 유전자 상의 변이를 검출하는 방법을 제공한다.In another embodiment of the present invention, 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.
본 발명의 프로브는 표적 유전자의 일부 서열과 특이적으로 혼성화할 수 있다. 상기 프로브의 일부 서열이 '타겟 영역' 이라 할 수 있다. 타겟 영역은 일예로 엑손 또는 엑손의 일부일 수 있다. 본 발명의 프로브는 타겟 영역에 상보적인 서열을 갖도록 제작되어, 혼성화, 어닐링 또는 증폭 조건 하에서 해당 타겟영역과 어닐링 또는 혼성화될 수 있다. 본 발명의 '혼성화'는 상보적인 단일가닥 핵산들이 이중-가닥 핵산을 형성하는 것을 의미한다. 혼성화는 2개의 핵산 가닥 간의 상보성이 완전할 경우(perfect match) 일어나거나 또는 일부 부정합(mismatch) 염기가 존재하여도 일어날 수 있다. 혼성화에 필요한 상보성의 정도는 혼성화 조건에 따라 달라질 수 있으며, 특히 온도에 의하여 조절될 수 있다.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.
상기 시료 핵산은 생물학적 시료로부터 분리된 DNA 또는 RNA일 수 있다. 예를 들면, 상기 생물학적 시료는 혈액, 타액, 뇨, 분변, 조직, 세포 및 생검물로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 시료는 보관된 생물학적 시료 또는 그로부터 분리된 핵산을 포함하는 것일 수 있다. 상기 보관은 알려진 방법에 의하여 보관된 것일 수 있다. 상기 핵산은 냉동 보관 또는 포르말린 고정된 파라핀 임베드된 조직을 상온에서 보관한 조직으로부터 유래된 것일 수 있다. 생물학적 시료로부터 핵산을 분리하는 방법은 잘 알려져 있다. 상기 시료는 환자의 세포, 조직, 기관, 체액으로부터 분리한 것일 수 있으며, 이 경우, 상기 시료는 통상적인 방법, 예를 들면, 관련 의학 기법에서 당업자에 의해 잘 공지된 방법을 이용하는 생검에 의해 수득될 수 있다.The sample nucleic acid may be DNA or RNA isolated from a biological sample. For example, 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.
상기 검출 방법에서, 상기 혼성화는 알려진 방법에 의하여 수행될 수 있다. 예를 들면, 핵산의 혼성화에 적절한 것으로 알려진 버퍼 중에서 상기 폴리뉴클레오티드와 시료 핵산을 인큐베이션함으로써 수행될 수 있다. 혼성화는 적절한 온도에서 수행될 수 있다. 혼성화에 적절한 온도는 예를 들면, 40 내지 80℃, 50 내지 75 ℃, 60 내지 70℃, 또는 62 내지 67℃일 수 있으며, 구체적으로는 65℃일 수 있다. 또한 혼성화 온도는 이에 제한되지 않고, 조성물에 포함된 폴리뉴클레오티드의 서열 및 길이에 따라 적절하게 선택될 수 있다. 혼성화 시간은 예를들면, 1 시간 내지 24시간 (밤새) 동안일 수 있다.In the detection method, 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. In addition, 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).
상기 핵산 서열의 확인은 예를 들면 시퀀싱(sequencing) 방법을 통해 확인할 수 있으며, 구체적으로는 차세대 염기서열분석법에 의해 확인할 수 있다. 용어 차세대 염기서열 분석법(next generation sequencing: NGS)은 전장유전체를 무수히 많은 조각을 분해하여 각 조각을 초병렬적으로 읽어낸 뒤 전산 기술을 이용하여 조합함으로써 방대한 유전체 정보를 빠르게 해독하는 방법이다. 차세대 염기서열 분석법에 의해 짧은 시간 내에 분석대상이 되는 시료에 대해 대량의 염기서열 데이터를 생성할 수 있다.The nucleic acid sequence can be confirmed by, for example, a sequencing method, and specifically, by a next-generation sequencing method. The term next generation sequencing (NGS) 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.
상기 초병렬 시퀀싱은 현재 차세대 염기서열 분석법으로 알려진 방법 및 장래 개발될 수 있는 방법을 포함한다. 상기 초병렬 시퀀싱은 합성에 의한 시퀀싱(sequencing by synthesis), 이온 토렌트(Ion-Torrent) 시퀀싱, 파이로시퀀싱(pyrosequencing), 라이게이션에 의한 시퀀싱, 나노포어 시퀀싱 및 단일-분자 실시간 시퀀싱으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것일 수 있다.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 one or more selected.
상기 프로브는 상기 타겟 영역과 특이적으로 혼성화될 수 있는 핵산 서열을 갖는, 예를 들면 75 내지 200개, 80 내지 200개, 90 내지 200개, 100 내지 200개, 100 내지 180개, 100 내지 160개, 100 내지 140개, 100 내지 120개의 크기의 핵산일 수 있다. 75개 이하의 크기를 가질 경우 타겟 영역에 대한 캡쳐 정확도가 낮으며, 200개 이상의 크기를 가질 경우 합성비용이 증가하는 단점을 갖는다.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.
본 발명의 프로브 세트는 타겟 시퀀싱(targeted sequencing)을 위한 타겟 캡쳐(target capture)에서 프로브로 이용될 수 있다. '타겟 시퀀싱'이란 전체 게놈 DNA가 아닌 게놈의 특정 영역(targeted region)만을 포획하여 분석하는 것으로, 다양한 유전자의 변이를 확인하는 대표적인 방법이다. 용어 '타겟 캡쳐'란, 시퀀싱 하기 전 특정 유전자 또는 기타 관심 부위를 DNA 라이브러리로부터 분리 및/또는 그 빈도를 증가시키기 위한 방법으로, 관심 부위는 시퀀싱을 위해 유지하고 나머지 물질을 제거한다. 특정 위치를 캡쳐하기 위해서는 프로브라는 기초 물질이 필요하다. 상기 프로브를 이용하여 유전체 DNA를 캡쳐하기 위해서는 이들 간의 상보적인 결합력을 이용하게 되는데 상보적 결합력은 DNA-DNA, RNA-DNA, RNA-RNA 순으로 강해지므로 효율적인 타겟 캡쳐(target capture)를 위해서 DNA 올리고뉴클레오티드의 형태로 합성된 프로브를 기내전사(in vitro transcription)를 통해 RNA 올리고뉴클레오티드의 형태(target capture RNA probe)를 만들어준다. 이후 혼성화 (hybridization) 과정을 통해 원하는 게놈(genome)상의 특정 타겟(target) 영역들만을 프로브를 이용하여 캡쳐하고, 캡쳐된 영역은 차세대 염기서열 분석기술(NGS) 을 이용하여 유전자 변이를 확인할 수 있다.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. In order to capture a specific location, a basic material called a probe is required. In order to capture genomic DNA using the probe, complementary binding force between them is used. Complementary binding strength is strengthened in the order of DNA-DNA, RNA-DNA, and RNA-RNA, so DNA 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). .
상기 검출 방법은 확인된 시료 핵산의 염기서열을 표준 염기서열과 비교하는 단계를 포함한다. 용어 표준 핵산 서열(reference nucleotide sequence)은 변이 확인을 위해 참조가 되는, 변이를 포함하지 않는 인간 유전자 서열을 의미할 수 있다. 예를 들면 표준 염기서열로서 미국 국립보건원 산하 생물공학정보연구소(NCBI)의 데이터베이스에 게시된 인간 유전자 염기서열, 구체적으로, NCBI37.1 또는 UCSC hg19(GRCh37)를 이용할 수 있다. 상기 시료 핵산의 염기서열과 표준 염기서열 간의 비교는 공지된 다양한 서열 비교 분석 프로그램, 예를 들면 Maq, Bowtie, SOAP, GSNAP 등을 이용하여 수행할 수 있다.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 (reference nucleotide sequence) may refer to a human gene sequence that does not contain a variation, which is referred to for identification. For example, as 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. 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.
상기 검출 방법은 시료 핵산의 변이를 확인하는 단계를 포함한다.The detection method includes the step of confirming the variation of the sample nucleic acid.
상기 변이 확인은 공지된 변이 검출 프로그램, 예를 들면 GATK, SAMtool, MoDIL, SeqSeq, PeMer, VariationHunter, Pindel, BreakDancer 및 Mutek 등을 이용하여 수행할 수 있으나, 이에 제한되지 않는다.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.
이하, 본 발명을 하기 실시예에 의해 더욱 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail by the following examples.
그러나, 이들 실시예는 본 발명에 대한 이해를 돕기 위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이들에 의해 제한되는 것은 아니다.However, these examples are only to aid understanding of the present invention, and the scope of the present invention is not limited by them in any sense.
실시예 1: 폐암과 관련된 유전자의 선정Example 1: Selection of genes related to lung cancer
폐암과 관련된 유전자로, AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1을 선정하였다.Genes related to lung cancer, AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 were selected.
실시예 2: 선정된 유전자에 대한 프로브 제작Example 2: Probe preparation for the selected gene
2.1 코딩 서열 확인2.1 Coding sequence identification
앞서 선정된 유전자들을 타겟으로 하여 해당 코딩 서열, 즉 단백질로써 코딩되는 엑손으로 구성된 부분에 대한 DNA 서열을 포함하도록 디자인하였다. 이때 UCSC genome browser를 이용해 모든 유전자의 전사체(transcript)를 모두 포함할 수 있도록 프로브를 구성하도록 하였다. 선정된 유전자들 중 ALK, BRAF, NTRK1, NTRK2, RET, ROS1 유전자에 대해서는 해당 유전자에서 fusion 유전자 변이가 발생하는 intron 위치를 선정하고, 이를 포획할 수 있는 프로브를 구성하였다.It 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. At this time, the probe was configured to include all transcripts of all genes using the UCSC genome browser. Among the selected genes, for the ALK, BRAF, NTRK1, NTRK2, RET, and ROS1 genes, intron positions in which the fusion gene mutation occurs in the corresponding genes were selected, and a probe capable of capturing them was constructed.
2.2. 프로브 서열 디자인2.2. Probe sequence design
유전자 별로 재조합된 서열의 CDS 시작지점을 기준으로 하여 레퍼런스 게놈 상에서 upstream 쪽으로 일정 영역(30-60bp) 확장하여 intergenic 영역에서 프로브 시작 지점을 설정하였다. 이로부터 프로브의 길이에 따라 일정 부분이 다음 순서의 것과 겹치도록 프로브를 순차적으로 디자인 진행하였다. 이때 이웃하는 프로브 간에 60bp 정도씩 겹치도록 하는 2x tiling 기법으로 디자인함으로써 모든 영역에 대하여 효율적으로 캡쳐되도록 하였다. 프로브의 on-target ratio 및 uniformity 향상을 위하여 양 말단에서 시작하는 terminal 프로브를 추가로 디자인해주었다. 프로브가 타겟 유전자와 결합하는 위치에 높은 GC ratio를 지니거나 특정 서열이 반복적으로 나타날 때 캡쳐 효율이 낮아질 수 있으므로 이를 보완하기 위하여 프로브 서열을 조정하는 방법(G 또는 C가 4개 이상 연속될 때에 4번째 염기를 A 또는 T로 치환하는 GC fix 과정)을 적용하여 효율적인 캡쳐가 되도록 하였다.Based on the CDS starting point of the recombined sequence for each gene, 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.
도 1은 프로브 디자인의 모식도를 나타낸다.1 shows a schematic diagram of a probe design.
2.3. rebalancing 과정2.3. rebalancing process
상기된 바와 같이 디자인된 프로브를 이용하여 타겟티드 시퀀싱 pilot test를 진행하여 캡쳐 결과를 확인한 후 캡쳐가 잘 되지 않은 부분은 프로브를 보충하며, 캡쳐가 많이 된 부분은 프로브 수를 감소시킴으로써 프로브 구성을 조정하는 rebalancing을 진행하였다. Normalized depth가 상대적으로 낮은 영역에 대해서는 동일한 프로브를 depth 수치에 따라 추가로 n배 삽입할 수 있으며, tiling depth를 높여주었다.After performing the targeted sequencing pilot test using the probe designed as described above, after confirming the capture result, 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.
또한, 캡쳐가 되지 않는 영역에 대해서는 타겟 서열에 GC가 높거나 특정 서열이 반복되어 캡쳐되지 않았다고 판단하여 주변부로 프로브를 30bp 이동하여 디자인함으로써 캡쳐의 효율을 올려주었다. Normalized depth가 상대적으로 높은 영역에 대해서는 tiling depth를 낮춰주거나, 해당 프로브의 개수를 줄여주었다. 이때 normalized depth가 높고 낮은 구간의 프로브 수에 따른 비율은 수배에서 수십 배까지 차이가 날 수 있으며 이는 절대적인 수치로 정해진 것이 아니고 상대적인 비율을 고려하여 조정하였다.In addition, for the non-capturing region, it was determined that GC was high in the target sequence or that a specific sequence was not repeated, and thus the probe was designed by moving the probe 30bp to the periphery, thereby increasing the efficiency of capture. For areas with relatively high normalized depth, the tiling depth was lowered or the number of probes was reduced. At this time, 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.
폐암과 관련된 유전자로 선정된 28개 유전자에 대한 프로브 제작 결과, 각 유전자에 대한 프로브 서열은 표 1에 기재된 바와 같다.As a result of probe production for 28 genes selected as genes related to lung cancer, probe sequences for each gene are shown in Table 1.
프로브 세트 번호Probe set number 유전자명Genetic name 프로브 서열번호Probe sequence number 프로브 세트 번호Probe set number 유전자명Genetic name 프로브 서열번호Probe sequence number
1One AKT1AKT1 2041 ~ 20442041 ~ 2044 1616 NF1NF1 2079 ~ 21132079 ~ 2113
22 ALKALK 203 ~ 249203 ~ 249 1717 NRASNRAS 166 ~ 174166-174
33 ARAFARAF 2337 ~ 23402337 ~ 2340 1818 NTRK1NTRK1 182 ~ 202182 ~ 202
44 ARID1AARID1A 91 ~ 16591 ~ 165 1919 NTRK2NTRK2 1373 ~ 18001373 ~ 1800
55 BRAFBRAF 1006 ~ 13461006 ~ 1346 2020 PIK3CAPIK3CA 431 ~ 440431 ~ 440
66 CBLCBL 1921 ~ 19291921 ~ 1929 2121 PTENPTEN 1895 ~ 19111895 ~ 1911
77 CDKN2ACDKN2A 1347 ~ 13721347 ~ 1372 2222 RB1RB1 1934 ~ 20401934 ~ 2040
88 EGFREGFR 745 ~ 885745 ~ 885 2323 RETRET 1801 ~ 18941801 ~ 1894
99 ERBB2ERBB2 2114 ~ 22462114 ~ 2246 2424 RIT1RIT1 175 ~ 181175 ~ 181
1010 HRASHRAS 1912 ~ 19201912 ~ 1920 2525 ROS1ROS1 441 ~ 744441 ~ 744
1111 KEAP1KEAP1 2291 ~ 23302291 ~ 2330 2626 SETD2SETD2 250 ~ 430250 ~ 430
1212 KRASKRAS 1930 ~ 19331930 ~ 1933 2727 STK11STK11 2247 ~ 22902247 ~ 2290
1313 MAP2K1MAP2K1 2045 ~ 20552045 ~ 2055 2828 TP53TP53 2056 ~ 20782056 ~ 2078
1414 METMET 886 ~ 1005886 ~ 1005 2929 U2AF1U2AF1 2331 ~ 23362331 ~ 2336
1515 MTORMTOR 1 ~ 901 to 90
실시예 3: 프로브의 캡쳐 효율 분석Example 3: Analysis of the capture efficiency of the probe
Human reference genome 샘플 3 가지 종류의 DNA를 이용하여 표준 DNA(Reference DNA)를 준비하고, 상기 실시예 2에서 디자인한 프로브 세트를 준비한 후 3 차례 유전자 포획 실험을 진행하여 패널의 유전자 포획 성능 및 안정성을 확인한 결과를 하기 표 2 및 3에 나타내었다.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.
이에 따르면, 3차례의 실험에서 모두 포획 대상 영역의 대부분을 성공적으로 획득할 수 있는 것을 확인하였으며, 패널의 포획 크기 대비 on-target 비율도 높은 성능을 나타내었다. 또한 세 번의 실험에서 성능의 편차가 거의 없는 것을 통해 패널이 안정적으로 동작하는 것을 확인하였다.According to this, in all three experiments, it was confirmed that most of the capture target area can be successfully obtained, and the on-target ratio to the capture size of the panel also showed high performance. In addition, in three experiments, it was confirmed that the panel was stably operated through little variation in performance.
Sample NameSample Name Total ReadTotal Read Total BaseTotal Base Mean Read SizeMean Read Size Target SizeTarget Size Mapping ratioMapping ratio Duplication ratioDuplication ratio On target ratioOn target ratio Mean depthMean depth
Reference DNA #1Reference DNA # 1 557,370557,370 81,706,79081,706,790 146.59146.59 115,944115,944 98.25%98.25% 2.64%2.64% 24.20%24.20% 149.06149.06
Reference DNA #2Reference DNA # 2 584,224584,224 85,729,96885,729,968 146.74146.74 115,944115,944 97.78%97.78% 2.21%2.21% 23.12%23.12% 148.25148.25
Reference DNA #3Reference DNA # 3 531,620531,620 77,810,44077,810,440 146.36146.36 115,944115,944 97.30%97.30% 2.22%2.22% 23.08%23.08% 134.47134.47
Sample NameSample Name CoverageCoverage
UncoveredUncovered >1X> 1X >10X> 10X >50X> 50X >100X> 100X
Reference DNA #1Reference DNA # 1 0.00009%0.00009% 99.9991%99.9991% 99.7068%99.7068% 95.0269%95.0269% 84.2683%84.2683%
Reference DNA #2Reference DNA # 2 0.0078%0.0078% 99.9922%99.9922% 99.5929%99.5929% 94.5577%94.5577% 83.9595%83.9595%
Reference DNA #3Reference DNA # 3 0.0181%0.0181% 99.9819%99.9819% 99.6412%99.6412% 93.4917%93.4917% 80.1249%80.1249%

Claims (12)

  1. 하기 (a), (b) 또는 (c)의 핵산 또는 그의 일부를 포함하는, 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브:Probes for detecting gene mutations that can cause lung cancer, comprising the nucleic acids of (a), (b) or (c) below:
    (a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1으로 이루어진 군으로부터 선택되는 하나 이상의 유전자를 구성하는 염기서열을 포함하는 핵산,(a) AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1 , ROS1, SETD2, STK11, TP53 and U2AF1 nucleic acid comprising a nucleotide sequence constituting one or more genes selected from the group consisting of,
    (b) 상기 (a)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(b) a nucleic acid comprising a base sequence complementary to the nucleic acid of (a) above,
    (c) 상기 (a)의 핵산, 또는 상기 (b)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산과 혼성화하고 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(c) A nucleic acid capable of hybridizing with a nucleic acid of (a) or a nucleic acid containing a nucleotide sequence complementary to the nucleic acid of (b) and detecting gene mutations that can cause lung cancer.
  2. 제1항에 있어서,According to claim 1,
    상기 (a)의 핵산이 AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1, RET, RIT1, ROS1, SETD2, STK11, TP53 및 U2AF1의 각 유전자를 구성하는 염기서열을 포함하는 핵산인 프로브.The nucleic acid of (a) is AKT1, ALK, ARAF, ARID1A, BRAF, CBL, CDKN2A, EGFR, ERBB2, HRAS, KEAP1, KRAS, MAP2K1, MET, MTOR, NF1, NRAS, NTRK1, NTRK2, PIK3CA, PTEN, RB1 , RET, RIT1, ROS1, SETD2, STK11, TP53 and U2AF1 are nucleic acid probes comprising nucleotide sequences constituting each gene.
  3. 하기 (d), (e) 또는 (f)의 핵산을 포함하는, 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 프로브:Probes for detecting genetic mutations that can cause lung cancer, comprising the nucleic acids of (d), (e) or (f):
    (d) 서열번호 1 내지 2340의 염기서열을 포함하는 핵산,(d) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 to 2340,
    (e) 서열번호 2341 내지 4680에 나타나는, 상기 (d)의 핵산에 대하여 상보적인 염기서열을 포함하는 핵산,(e) a nucleic acid comprising a nucleotide sequence complementary to the nucleic acid of (d) shown in SEQ ID NOs: 2341 to 4680,
    (f) 상기 (d) 또는 상기 (e)의 핵산의 염기서열에 대하여 70% 이상의 상동성을 갖는 염기서열을 포함하고, 폐암을 유발할 수 있는 유전자 변이를 검출할 수 있는 핵산.(f) A nucleic acid capable of detecting a gene mutation capable of causing lung cancer, comprising a base sequence having 70% or more homology to the base sequence of the nucleic acid of (d) or (e).
  4. 제1 항 내지 제3 항 중 어느 한 항에 따른 프로브를 제조하되, 상기 프로브는 단백질로 코딩되는 엑손(exon) 부분의 DNA 시퀀스(coding sequence) 및 특정 융합(fusion) 유전자 변이가 발생하는 인트론(intron) 부분의 DNA 시퀀스를 포함하도록 시퀀스 디자인된 것인 프로브의 제조방법.The probe according to any one of claims 1 to 3, wherein the probe is an intron that generates a DNA sequence (coding sequence) and a specific fusion gene mutation of an exon portion encoded by a protein ( intron) A method of manufacturing a probe that is sequence designed to include a DNA sequence of a part.
  5. 제4 항에 있어서,According to claim 4,
    상기 프로브의 시퀀스 디자인은 타겟 영역의 모든 염기 및 타겟 영역 밖의 일부를 포함하도록 디자인되되, 2X 타일링 방법에 의한 디자인과 타겟의 양 말단에서 시작하는 터미널 프로브의 추가 디자인을 포함하는 것인 프로브의 제조방법.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. .
  6. 제4 항에 있어서,According to claim 4,
    상기 시퀀스는 GC fix 과정을 거쳐 조정된 것인 프로브의 제조방법.The sequence is a method of manufacturing a probe that is adjusted through a GC fix process.
  7. 제4 항에 있어서,According to claim 4,
    상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율 보다 낮은 부분에, 해당 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것인 프로브의 제조방법.The method of manufacturing a probe further comprising a step of re-balancing by adding a corresponding probe to a portion where hybridization is lower than an average hybridization rate by measuring the hybridization rate of the probe.
  8. 제4 항에 있어서,According to claim 4,
    상기 프로브의 혼성화율(hybridization rate)을 측정하여 혼성화가 평균 혼성화율 보다 높은 부분에, 해당 프로브를 제외한 나머지 프로브를 추가 삽입하여 재조정(re-balancing) 단계를 더 포함하는 것인 프로브의 제조방법.The method of preparing a probe further comprising the step of re-balancing by measuring the hybridization rate of the probe and inserting the remaining probes except the corresponding probe into the portion where hybridization is higher than the average hybridization rate.
  9. 제1 항 내지 제3 항 중 어느 한 항에 따른 프로브를 포함하는 폐암을 유발할 수 있는 유전자 변이를 검출하기 위한 키트.A kit for detecting a gene mutation that can cause lung cancer, comprising the probe according to any one of claims 1 to 3.
  10. 제1 항 내지 제3 항 중 어느 한 항에 따른 프로브와 시료 핵산을 접촉시켜, 상기 시료 핵산과 상기 프로브의 혼성화 산물을 얻는 단계;Contacting the probe according to any one of claims 1 to 3 with a sample nucleic acid to obtain a hybridization product of the sample nucleic acid and the probe;
    상기 혼성화 산물 중의 상기 시료 핵산의 서열을 확인하는 단계; 및Identifying the sequence of the sample nucleic acid in the hybridization product; And
    상기 시료 핵산의 확인된 핵산 서열을 표준 핵산 서열과 비교하여 상기 시료 핵산의 변이를 확인하는 단계를 포함하는 폐암을 유발할 수 있는 유전자 상의 변이를 검출하는 방법.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.
  11. 제10 항에 있어서,The method of claim 10,
    상기 시료 핵산의 서열의 확인은 초병렬 시퀀싱에 의해 분석되는 것인 방법.The method of confirming the sequence of the sample nucleic acid is analyzed by super-parallel sequencing.
  12. 제11 항에 있어서,The method of claim 11,
    상기 초병렬 시퀀싱은 합성에 의한 시퀀싱, 이온 토렌트 시퀀싱, 파이로시퀀싱, 라이게이션에 의한 시퀀싱, 나노포어 시퀀싱 및 단일-분자 실시간 시퀀싱으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인 방법.The super-parallel sequencing is one or more methods selected from the group consisting of synthetic sequencing, ion torrent sequencing, pyrosequencing, ligation sequencing, nanopore sequencing, and single-molecule real-time sequencing.
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