WO2022139062A1 - Method, kit and nucleic acid composition for detecting gene methylation for early diagnosis of lung cancer - Google Patents
Method, kit and nucleic acid composition for detecting gene methylation for early diagnosis of lung cancer Download PDFInfo
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- 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
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
Definitions
- the present invention relates to a method for detecting methylation of a gene for early diagnosis of lung cancer.
- the present invention relates to a primer or probe for detecting a methylated lung cancer target gene.
- the present invention relates to a composition for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene.
- the present invention relates to a primer or probe for detecting a methylated lung cancer target gene, or a kit for early diagnosis of lung cancer comprising the composition.
- DNA methylation plays a role in protecting against invasion of foreign genes in prokaryotes, and plays an important role in regulating gene expression during development in eukaryotes. Recent studies have revealed that DNA methylation is also involved in genome imprinting and stabilization, inactivation of the X chromosome, etc., and affects tissue-specific gene activity and increase or suppression of the expression of disease-related genes.
- cancer occurs when the CpG island of a tumor suppressor gene is methylated and its expression is blocked. Since DNA methylation is a representative phenomenon that occurs at the earliest stage of cancer development, it is recognized as an optimal tool for early diagnosis of cancer. Recently, for the early diagnosis and screening of cancer, the development of a diagnostic technology using the methylation of a specific gene as a biomarker is being actively studied. In order to detect CpG island methylation associated with these biomarker genes, methods such as bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and pyrosequencing and methylation-specific polymerase chain reaction (PCR) are being actively tried.
- COBRA combined bisulfite restriction analysis
- PCR methylation-specific polymerase chain reaction
- PNA was first reported in 1991 as DNA-like DNA in which nucleotides are linked by peptide bonds rather than phosphate bonds. PNA causes a hybridization reaction with a natural nucleic acid of a complementary base sequence to form a double strand. When the number of nucleic acid bases is the same, PNA/DNA double strands are more stable than DNA/DNA double strands, and PNA/RNA double strands are more stable than DNA/RNA double strands. As the basic backbone of PNA, one in which N-(2-aminoethyl)glycine is repeatedly linked by an amide bond is most commonly used. is electrically neutral.
- PNA nucleobases present in PNA occupy a space similar to that of nucleic acid bases of DNA, and the distance between nucleic acid bases is almost the same as that of natural nucleic acids.
- PNA is not only chemically more stable than natural nucleic acids, but also biologically stable because it is not degraded by nucleases or proteases. Since PNA is also electrically neutral, the stability of PNA/DNA and PNA/RNA double strands is not affected by salt concentration. Because of this property, PNA can recognize complementary nucleic acid sequences better than natural nucleic acids, and thus has applications for diagnostic or other biological and medical purposes.
- lung cancer can be diagnosed early by detecting methylation of a biomarker gene through a PNA probe, and completed the present invention.
- Patent Document 1 Republic of Korea Patent Publication No. 10-2011-0130638 (2011.12.06.)
- Non-Patent Document 1 Nielsen et al., Science, 254, pp. 1497-1500, 1991
- LDCT Low-dose chest CT
- CT computed tomography
- biopsy to confirm lung cancer due to a high false-positive rate such as misdiagnosing lung nodules as lung cancer or errors depending on the inspector's perspective.
- CT computed tomography
- patients have psychological anxiety and complications caused by invasive biopsy, problems with high-level radiation coverage, and burdens for high examination and treatment costs, and doctors have various problems such as overtreatment and problems due to overdiagnosis.
- An object of the present invention is to provide a method for detecting methylation of a gene for early diagnosis of lung cancer using a PNA probe.
- An object of the present invention is to provide a kit for early diagnosis of lung cancer by analyzing whether or not a gene is methylated, including the PNA probe of the present invention.
- the present invention provides a method comprising: amplifying a target gene in the presence of a PNA probe capable of hybridizing with a methylated lung cancer target gene; amplifying the unmethylated gene in the presence of a PNA probe capable of hybridizing with the unmethylated gene; and determining whether the target gene is methylated by analyzing the melting curve of the amplification product.
- the present invention also relates to a primer or probe for detecting a methylated lung cancer target gene, comprising at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
- the present invention also relates to a composition for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene.
- the present invention further relates to a kit for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the present invention further relates to a method for early diagnosis of lung cancer comprising treating a sample with the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the present invention further relates to a diagnostic use of the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the present invention it is possible to determine whether or not lung cancer is present through gene methylation analysis of a biomarker for early diagnosis of lung cancer. Specifically, amplifying the target gene in the presence of a PNA probe capable of hybridizing with the methylated lung cancer target gene; amplifying the unmethylated gene in the presence of a PNA probe capable of hybridizing with the unmethylated gene; and determining whether the target gene is methylated by analyzing the melting curve of the amplification product.
- MPR methylation ratio
- ⁇ Ct (Ct value of unmethylated gene - Ct value of methylated gene)
- ⁇ Ct ( ⁇ Ct of gene sample without probe addition- ⁇ Ct of gene sample with probe added)
- ⁇ Tm kXmCXn formula (1 ) [wherein k is a constant, mC is the difference between Tm when there is one methylated cytosine, and n is the number of methylated cytosines].
- At least one of ⁇ Tm and ⁇ Ct values in the PCR performed in the above step is measured.
- the Tm value or Ct value of the methylated gene is measured to be high, and the value of ⁇ Tm obtained by subtracting the Tm value of the gene without methylation from the Tm value of the methylated gene or the methylation value from the Ct value of the methylated DNA Whether or not the target DNA is methylated can be determined by checking the value of ⁇ Ct by subtracting the Ct value of the DNA in which the methylation does not occur.
- ⁇ Ct cycle threshold
- the ⁇ Ct is ( ⁇ Ct of a gene sample without probe addition- ⁇ Ct of a gene sample with a reduction probe added), and ⁇ Ct is (Ct value of unmethylated gene - Ct value of methylated gene).
- the base sequence of the target gene according to an embodiment of the present invention is a base methylated with any one or a combination of two or more of a methylated base sequence, a hydroxymethylated base sequence, a formyl methylated base sequence, and a carboxyl methylated base sequence It may have a sequence.
- ⁇ Tm according to an embodiment of the present invention is characterized in that it satisfies Equation (1) below.
- k is a constant
- mC is the difference in Tm when there is one methylated cytosine
- n is the number of methylated cytosines.
- the ⁇ Tm may be 2°C or higher and 5°C or lower when one methylated cytosine is used.
- the detection method according to an embodiment of the present invention is standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), quantitative PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization) selected from It can be any one.
- the detection method may include a method capable of detecting gene amplification efficiency or a PCR product.
- a method capable of detecting gene amplification efficiency or a PCR product Preferably, real-time PCR may be used, but it is not necessarily limited thereto, and the PCR may be used without limitation as long as it is used in the art.
- the probe may be, for example, a reduction probe.
- the reduction probe (inhibition probe; reduction probe) may be a probe that binds to a methylated detection site and inhibits gene amplification. It is possible to induce a difference in amplification by binding more strongly to the methylated detection site than to the unmethylated detection site.
- the methylated cytosine and the reduction probe form complementary binding.
- Complementary bonding is to form a strong intermolecular attraction by forming three hydrogen bonds between the methylated cytosine and the guanine base of the reduction probe.
- Methylated cytosine refers to a cytosine that is methylated at the 5th carbon of the pyrimidine ring. Since the methyl group corresponds to an electron donation group (EDG), the methylated cytosine is a complementary base, guanine, and stronger hydrogen. make a bond An increase in the intermolecular interaction of guanine and methylated cytosine is associated with an increase in the electron-donating properties of the C5-substituent. Also, only pairs containing 5-methyl cytosine bind more strongly than the guanine and cytosine pairs formed by standard cytosines. As a result, when PCR is performed, the Tm between the methylated DNA and the reduction probe has a higher value than the Tm between the unmethylated DNA and the reduction probe.
- EDG electron donation group
- At least one of ⁇ Tm and ⁇ Ct values in the PCR performed in the above step is measured.
- the Tm value or Ct value of the methylated gene is measured to be high, and the value of ⁇ Tm obtained by subtracting the Tm value of the gene without methylation from the Tm value of the methylated gene or the methylation value from the Ct value of the methylated DNA Whether or not the target DNA is methylated can be determined by checking the value of ⁇ Ct by subtracting the Ct value of the DNA in which the methylation does not occur.
- a method for detecting methylation of a gene using a reduction probe that determines the degree (X) of methylation by specifying a ⁇ Ct (cycle threshold) value.
- the ⁇ Ct is ( ⁇ Ct of the gene sample without the addition of the reduction probe- ⁇ Ct of the gene sample with the addition of the reduction probe), and ⁇ Ct is (Ct value of unmethylated gene - Ct value of methylated gene).
- the Ct value is high.
- the methylated DNA has a value of ⁇ Ct>0 and ⁇ Ct ⁇ 0.
- the degree of methylation may represent a value between 0 and 1. The closer to 1, the lower the methylation degree, and the closer to 0, the higher the methylation degree.
- the primers and probes used for the gene amplification may include one or more selected from the group consisting of SEQ ID NOs: 1 to 30.
- the present invention also relates to a primer or probe for detecting a methylated lung cancer target gene, comprising at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
- the present invention also relates to a composition for early diagnosis of lung cancer comprising a primer or probe for detecting a methylated lung cancer target gene.
- the present invention further relates to a method for early diagnosis of lung cancer comprising treating a sample with the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the present invention further relates to a diagnostic use of the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the present invention further relates to a kit for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene, or the composition.
- the kit comprises a compartmentalized carrier means for holding a sample, a first container capable of containing template DNA, a second container containing a primer complementary to a 5'-CpG-3' sequence region for amplifying the sample, and an amplified product one or more vessels comprising a third vessel containing a probe for detecting
- the carrier means is suitable for containing one or more containers, such as bottles and tubes, each container containing the independent components used in the method of the present invention.
- containers such as bottles and tubes
- each container containing the independent components used in the method of the present invention.
- one of ordinary skill in the art can readily dispense the required formulation in a container.
- Example 1 Selection of gene methylation markers for early diagnosis of lung cancer
- Primers and probes for PCR amplification were prepared to check the methylation of the five selected genes, and the final selection was made as follows through optimization.
- Human HCT116 DKO Methylated DNA was used as the standard for methylation determination, and the detection concentrations were 0.5, 1, 2.5, 5, 10, and 15 ng/ul, and each concentration was repeated 5 times.
- the minimum detection limit judgment criterion was set to a coefficient of variation (%) less than 5, and the minimum detection limit according to the concentration was confirmed to be 10ng.
- Example 4 Minimum detection limit according to standard methylation ratio (%)
- Human HCT116 DKO Methylated DNA was used as a standard for determining methylation, and methylation rates were 0.5, 1, 5, 10, 50, and 100%, and each concentration was repeated 5 times.
- the minimum detection limit judgment criterion was set to a coefficient of variation (%) less than 5, and the minimum detection limit according to the methylation ratio was confirmed to be 1%.
- Human HCT116 DKO Methylated DNA was used as the standard for methylation determination, and 3 lots, 3 equipment, and 3 testers performed 3 repetitions for each experiment, a total of 81 repetitions were performed.
- the reproducibility criterion was set to a coefficient of variation (%) less than 5, and as a result, it was confirmed to be less than CV 5.
- MPR methylation ratio
- LDCT low-dose chest CT
Abstract
The present invention relates to a method for detecting gene methylation for an early diagnosis of lung cancer, a primer or probe for detecting lung cancer targeted gene, and a composition and kit for an early diagnosis of lung cancer. The present invention can detect gene methylation using a PNA probe without a bisulfite treatment, and thus enables detection with high sensitivity and accuracy and is useful in diagnosing early stage lung cancer.
Description
본 발명은 폐암 조기진단을 위한 유전자의 메틸화 검출 방법에 관한 것이다. The present invention relates to a method for detecting methylation of a gene for early diagnosis of lung cancer.
본 발명은 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브에 관한 것이다.The present invention relates to a primer or probe for detecting a methylated lung cancer target gene.
본 발명은 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브를 포함하는 폐암 조기 진단용 조성물에 관한 것이다. The present invention relates to a composition for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene.
본 발명은 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기의 조성물을 포함하는 폐암 조기 진단용 키트에 관한 것이다. The present invention relates to a primer or probe for detecting a methylated lung cancer target gene, or a kit for early diagnosis of lung cancer comprising the composition.
DNA 메틸화는 원핵생물에서는 외부 유전자의 침입으로부터 보호해 주는 역할을 하며, 진핵생물에서는 발달과정에서 유전자의 발현을 조절해주는 중요한 역할을 하는 것으로 알려져 있다. 최근의 연구에서는 DNA 메틸화가 그 외에도 유전체 각인 및 안정화나 X염색체의 불활성화 등에도 관여하며, 조직특이적 유전자 활성, 질병과 연관된 유전자들의 발현 증가 또는 억제에도 영향을 미친다는 것이 밝혀지고 있다.It is known that DNA methylation plays a role in protecting against invasion of foreign genes in prokaryotes, and plays an important role in regulating gene expression during development in eukaryotes. Recent studies have revealed that DNA methylation is also involved in genome imprinting and stabilization, inactivation of the X chromosome, etc., and affects tissue-specific gene activity and increase or suppression of the expression of disease-related genes.
포유류 세포의 게놈 DNA에는 A, C, G, T 외에 5번째 염기가 존재하며, 이는 시토신 고리의 5번째 탄소에 메틸기 가 붙은 5-메틸시토신(5-mC)이다. 5-mC는 항상 CG 다이뉴클레오타이드의 C에만 오며(5'-mCG-3'), 이러한 CG를 흔히 CpG라고 표시한다. CpG의 C는 대부분이 메틸기가 붙어서 메틸화되어 있다. 이러한 메틸화 현상은 암을 포함한 다양한 질병의 원인으로 알려져 있으며, CpG섬의 메틸화에 의해 해당하는 유전자의 발현이 억제된다고 알려져 있다. In the genomic DNA of mammalian cells, there is a 5th base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) with a methyl group attached to the 5th carbon of the cytosine ring. 5-mC always occurs only at C of a CG dinucleotide (5'-mCG-3'), and this CG is often denoted as CpG. Most of C in CpG is methylated with a methyl group attached. This methylation phenomenon is known to be the cause of various diseases including cancer, and it is known that the expression of the corresponding gene is suppressed by methylation of the CpG island.
특히, 종양 억제 유전자의 CpG섬이 메틸화되어 발현이 차단됨으로써 암이 발생하는 것으로 알려져 있다. DNA 메틸화 현상은 암 발생의 가장 초기에 일어나는 대표적인 현상이기 때문에 암 조기진단을 위한 최적의 도구로 인식되고 있다. 최근에는, 암 조기진단 및 스크리닝을 위해서 특정 유전자의 메틸화 현상을 바이오마커로 이용한 진단기술의 개발이 활발히 연구되고 있다. 이러한 바이오마커 유전자와 연관된 CpG섬 메틸화를 검출하기 위하여 bisulfite sequencing, combined bisulfite restriction analysis(COBRA), 그리고 pyrosequencing과 메틸화특이 PCR(polymerase chain reaction) 등과 같은 방법들이 적극적으로 시도되고 있다.In particular, it is known that cancer occurs when the CpG island of a tumor suppressor gene is methylated and its expression is blocked. Since DNA methylation is a representative phenomenon that occurs at the earliest stage of cancer development, it is recognized as an optimal tool for early diagnosis of cancer. Recently, for the early diagnosis and screening of cancer, the development of a diagnostic technology using the methylation of a specific gene as a biomarker is being actively studied. In order to detect CpG island methylation associated with these biomarker genes, methods such as bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and pyrosequencing and methylation-specific polymerase chain reaction (PCR) are being actively tried.
PNA는 핵산염기가 인산 결합이 아니라 펩티드 결합으로 연결된 유사 DNA로 1991년에 처음 보고되었다. PNA는 상보적인 염기 서열의 천연 핵산과 혼성화(hybridization) 반응을 일으켜서 겹가닥을 형성한다. 핵산 염기의 수가 같은 경우 PNA/DNA 겹가닥은 DNA/DNA 겹가닥보다, PNA/RNA 겹가닥은 DNA/RNA 겹가닥보다 안정하다. PNA의 기본 골격으로는 N-(2-아미노에틸)글리신이 아미드 결합에 의해 반복적으로 연결된 것이 가장 흔히 쓰이고, 이 경우 펩티드 핵산의 기본 골격(backbone)은 음전하를 띠는 천연 핵산의 기본 골격과 달리 전기적으로 중성이다. PNA에 존재하는 4개의 핵산염기(nucleobase)는 DNA의 핵산 염기와 비슷한 공간을 차지하고 핵산 염기 사이의 거리도 천연 핵산의 경우와 거의 같다. PNA는 화학적으로 천연 핵산보다 안정할 뿐 아니라 핵산분해효소(nuclease) 나 단백질분해효소(protease)에 의해 분해되지 않아 생물학적으로도 안정하다. PNA는 또한 전기적으로 중성이기 때문에 PNA/DNA, PNA/RNA 겹가닥의 안정성은 염 농도에 영향을 받지 않는다. 이러한 성질 때문에 PNA는 상보적인 핵산 염기 서열을 천연 핵산보다 더 잘 인식할 수 있어서 진단 또는 다른 생물학적, 의학적 목적으로 응용된다.PNA was first reported in 1991 as DNA-like DNA in which nucleotides are linked by peptide bonds rather than phosphate bonds. PNA causes a hybridization reaction with a natural nucleic acid of a complementary base sequence to form a double strand. When the number of nucleic acid bases is the same, PNA/DNA double strands are more stable than DNA/DNA double strands, and PNA/RNA double strands are more stable than DNA/RNA double strands. As the basic backbone of PNA, one in which N-(2-aminoethyl)glycine is repeatedly linked by an amide bond is most commonly used. is electrically neutral. Four nucleobases present in PNA occupy a space similar to that of nucleic acid bases of DNA, and the distance between nucleic acid bases is almost the same as that of natural nucleic acids. PNA is not only chemically more stable than natural nucleic acids, but also biologically stable because it is not degraded by nucleases or proteases. Since PNA is also electrically neutral, the stability of PNA/DNA and PNA/RNA double strands is not affected by salt concentration. Because of this property, PNA can recognize complementary nucleic acid sequences better than natural nucleic acids, and thus has applications for diagnostic or other biological and medical purposes.
이러한 기술적 배경하에서, 본 출원의 발명자들은 PNA 프로브를 통해 바이오마커 유전자의 메틸화 검출을 통해 폐암을 조기 진단할 수 있음을 확인하고, 본 발명을 완성하였다. Under this technical background, the inventors of the present application confirmed that lung cancer can be diagnosed early by detecting methylation of a biomarker gene through a PNA probe, and completed the present invention.
선행기술문헌Prior art literature
(특허문헌 1) 대한민국 공개특허공보 10-2011-0130638호 (2011.12.06.)(Patent Document 1) Republic of Korea Patent Publication No. 10-2011-0130638 (2011.12.06.)
(비특허문헌 1) Nielsen et al., Science, 254, pp. 1497-1500, 1991(Non-Patent Document 1) Nielsen et al., Science, 254, pp. 1497-1500, 1991
발명의 요약Summary of the invention
현행 폐암 조기진단 권고법인 저선량 흉부 CT (LDCT)는 폐결절을 폐암으로 오진하거나, 검사자의 시각에 따른 오류 등 높은 위양성율로 인해 폐암 확진을 위해 컴퓨터 단층촬영(Computed Tomography, CT)나 조직검사와 같은 추가 시험이 필요한 한계를 가지고 있다. 이로 인해 환자들은 침습적인 조직검사에 따른 심리적 불안감과 합병증 발생, 고준위 방사선 피복의 문제, 높은 검진 및 치료비에 대한 부담을 가지게 되며, 의사들은 과진료, 과진단으로 인한 문제 등 다양한 문제점을 가지고 있다. Low-dose chest CT (LDCT), which is currently recommended for early diagnosis of lung cancer, uses additional methods such as computed tomography (CT) or biopsy to confirm lung cancer due to a high false-positive rate such as misdiagnosing lung nodules as lung cancer or errors depending on the inspector's perspective. It has limitations that require testing. As a result, patients have psychological anxiety and complications caused by invasive biopsy, problems with high-level radiation coverage, and burdens for high examination and treatment costs, and doctors have various problems such as overtreatment and problems due to overdiagnosis.
따라서, LDCT을 통해 폐결절과 폐암의 분별의 위양성율은 낮추고, 정확도 높은 진단법 개발이 필요한 상황이다. 또한 혈액과 같은 비침습적인 방법으로 채취할 수 있는 시료 내의 유전자 바이오 마커 기반 분자 진단법 개발을 하고자 한다.Therefore, it is necessary to develop a diagnostic method with high accuracy and lowering the false-positive rate for the classification of lung nodules and lung cancer through LDCT. In addition, we intend to develop a molecular diagnostic method based on genetic biomarkers in samples that can be collected by non-invasive methods such as blood.
본 발명의 목적은 PNA 프로브를 이용하여 폐암 조기진단을 위한 유전자의 메틸화 검출 방법을 제공하기 위한 것이다.An object of the present invention is to provide a method for detecting methylation of a gene for early diagnosis of lung cancer using a PNA probe.
본 발명의 목적은 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브를 제공하기 위한 것이다. It is an object of the present invention to provide a primer or probe for detecting a methylated lung cancer target gene.
본 발명의 목적은 PNA 프로브를 포함하는 폐암 조기 진단용 조성물을 제공하기 위한 것이다. It is an object of the present invention to provide a composition for early diagnosis of lung cancer comprising a PNA probe.
본 발명의 PNA 프로브를 포함하는, 유전자의 메틸화 여부 분석을 통한 폐암 조기 진단 키트를 제공하기 위한 것이다. An object of the present invention is to provide a kit for early diagnosis of lung cancer by analyzing whether or not a gene is methylated, including the PNA probe of the present invention.
상기 목적을 달성하기 위하여, 본 발명은 메틸화된 폐암 표적 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 표적 유전자를 증폭시키는 단계; 비메틸화 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 메틸화되지 않은 유전자를 증폭시키는 단계; 및 상기 증폭 산물의 융해곡선을 분석하여 표적 유전자의 메틸화 여부를 판별하는 단계를 포함하는, 폐암 조기진단을 위한 유전자의 메틸화 검출 방법에 관한 것이다.In order to achieve the above object, the present invention provides a method comprising: amplifying a target gene in the presence of a PNA probe capable of hybridizing with a methylated lung cancer target gene; amplifying the unmethylated gene in the presence of a PNA probe capable of hybridizing with the unmethylated gene; and determining whether the target gene is methylated by analyzing the melting curve of the amplification product.
본 발명은 또한, 서열번호 1 내지 서열번호 30으로 구성된 군에서 선택되는 하나 이상을 포함하는, 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브에 관한 것이다.The present invention also relates to a primer or probe for detecting a methylated lung cancer target gene, comprising at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
본 발명은 또한, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브를 포함하는 폐암 조기 진단용 조성물에 관한 것이다.The present invention also relates to a composition for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물을 포함하는 폐암 조기 진단용 키트에 관한 것이다.The present invention further relates to a kit for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene, or the composition.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물을 검체에 처리하는 단계를 포함하는 폐암 조기 진단방법에 관한 것이다.The present invention further relates to a method for early diagnosis of lung cancer comprising treating a sample with the primer or probe for detecting the methylated lung cancer target gene, or the composition.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물의 진단 용도에 관한 것이다. The present invention further relates to a diagnostic use of the primer or probe for detecting the methylated lung cancer target gene, or the composition.
도 1은 본 발명에 따른 메틸화 여부 분석에 대한 실시간 중합효소연쇄반응 결과를 나타낸 것이다.1 shows the results of real-time polymerase chain reaction for methylation analysis according to the present invention.
도 2는 본 발명에 대한 검증으로 기존 파이로시퀀싱 (Pyrosequencing)으로 메틸화 분석한 결과이다. 2 is a result of methylation analysis by conventional pyrosequencing as a verification for the present invention.
발명의 상세한 설명 및 구체적인 구현예DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS OF THE INVENTION
이하에서 본 발명에 대하여 구체적으로 설명한다. 본 명세서에서 사용되는 용어는 따로 정의하지 않는 경우 해당 분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 내용으로 해석되어야 할 것이다. 본 명세서의 도면 및 실시예는 통상의 지식을 가진 자가 본 발명을 쉽게 이해하고 실시하기 위한 것으로 도면 및 실시예에서 발명의 요지를 흐릴 수 있는 내용은 생략될 수 있으며, 본 발명이 도면 및 실시예로 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those of ordinary skill in the art to easily understand and practice the present invention, and contents that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments is not limited to
본 발명은 폐암 조기진단 바이오마커의 유전자 메틸화 분석을 통해 폐암 여부를 판별할 수 있다. 구체적으로, 메틸화된 폐암 표적 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 표적 유전자를 증폭시키는 단계; 비메틸화 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 메틸화되지 않은 유전자를 증폭시키는 단계; 및 상기 증폭 산물의 융해곡선을 분석하여 표적 유전자의 메틸화 여부를 판별하는 단계를 포함하는, 폐암 조기진단을 위한 유전자의 메틸화 검출 방법에 관한 것이다.In the present invention, it is possible to determine whether or not lung cancer is present through gene methylation analysis of a biomarker for early diagnosis of lung cancer. Specifically, amplifying the target gene in the presence of a PNA probe capable of hybridizing with the methylated lung cancer target gene; amplifying the unmethylated gene in the presence of a PNA probe capable of hybridizing with the unmethylated gene; and determining whether the target gene is methylated by analyzing the melting curve of the amplification product.
상기 메틸화 검출 방법은 유전자의 증폭 없이, 메틸화된 또는 비메틸화 유전자와 프로브의 ΔTm (melting temperature) 및/또는 ΔCt(cycle threshold) 값을 측정하여 메틸화 여부 또는 메틸화 정도를 분석하는 단계를 포함할 수 있다. ΔTm 또는 ΔCt의 수치를 통해 유전자의 메틸화 여부를 판단하거나, 또는 ΔΔCt값을 특정하여 MPR=2 ΔΔCt를 통해 메틸화 비율(MPR)을 계산할 수 있다. The methylation detection method may include analyzing whether methylation or the degree of methylation is methylated by measuring ΔTm (melting temperature) and/or ΔCt (cycle threshold) values of a methylated or unmethylated gene and a probe without amplification of the gene. . Whether or not a gene is methylated can be determined based on the value of ΔTm or ΔCt, or the methylation ratio (MPR) can be calculated through MPR=2 ΔΔCt by specifying the ΔΔCt value.
이 때, ΔCt=(메틸화되지 않은 유전자의 Ct값-메틸화된 유전자의 Ct값), ΔΔCt=(프로브의 첨가 없는 유전자 시료의 ΔCt-프로브를 첨가한 유전자 시료의 ΔCt), ΔTm=kXmCXn 식 (1) [상기 식에서 k는 상수, mC는 메틸화된 시토신이 1개일 경우의 Tm의 차이, n은 메틸화된 시토신의 개수이다.]로 계산될 수 있다. In this case, ΔCt=(Ct value of unmethylated gene - Ct value of methylated gene), ΔΔCt=(ΔCt of gene sample without probe addition-ΔCt of gene sample with probe added), ΔTm=kXmCXn formula (1 ) [wherein k is a constant, mC is the difference between Tm when there is one methylated cytosine, and n is the number of methylated cytosines].
상기 단계에서 수행된 PCR에서의 ΔTm과 ΔCt값 중 적어도 하나 이상을 측정한다. 메틸화된 시토신을 가진 표적 DNA의 경우 비메틸화된 DNA에 비하여 프로브와 더 강한 결합을 함으로써 PCR 수행시 증폭 억제 효과가 나타난다. 이를 이용하여 메틸화가 일어난 유전자의 Tm값 또는 Ct값이 높게 측정이 되고, 메틸화가 일어난 유전자의 Tm값에서 메틸화가 일어나지 않은 유전자의 Tm값을 뺀 ΔTm의 수치 또는 메틸화가 일어난 DNA의 Ct값에서 메틸화가 일어나지 않은 DNA의 Ct값을 뺀 ΔCt의 수치를 확인함으로써 표적 DNA의 메틸화 여부를 판단할 수 있다. At least one of ΔTm and ΔCt values in the PCR performed in the above step is measured. In the case of target DNA having methylated cytosine, the amplification inhibitory effect appears during PCR by stronger binding to the probe than that of unmethylated DNA. Using this, the Tm value or Ct value of the methylated gene is measured to be high, and the value of ΔTm obtained by subtracting the Tm value of the gene without methylation from the Tm value of the methylated gene or the methylation value from the Ct value of the methylated DNA Whether or not the target DNA is methylated can be determined by checking the value of ΔCt by subtracting the Ct value of the DNA in which the methylation does not occur.
ΔΔCt(cycle threshold)값을 특정하여 메틸화의 정도(X)를 판단하는 프로브를 이용한 유전자의 메틸화 검출 방법을 제공한다. 상기 ΔΔCt는 (프로브의 첨가 없는 유전자 시료의 ΔCt-리덕션 프로브를 첨가한 유전자 시료의 ΔCt)이고, ΔCt는(메틸화되지 않은 유전자의 Ct값-메틸화된 유전자의 Ct값)을 나타낸다.Provided is a method for detecting methylation of a gene using a probe for determining the degree (X) of methylation by specifying a ΔΔCt (cycle threshold) value. The ΔΔCt is (ΔCt of a gene sample without probe addition-ΔCt of a gene sample with a reduction probe added), and ΔCt is (Ct value of unmethylated gene - Ct value of methylated gene).
본 발명의 일 예에 따른 상기 표적 유전자의 염기서열은 메틸화된 염기서열, 하이드록시 메틸화된 염기서열, 포르밀 메틸화된 염기서열, 카르복실 메틸화된 염기서열 중 어느 하나 또는 2이상의 조합으로 메틸화된 염기서열을 가지는 것일 수 있다.The base sequence of the target gene according to an embodiment of the present invention is a base methylated with any one or a combination of two or more of a methylated base sequence, a hydroxymethylated base sequence, a formyl methylated base sequence, and a carboxyl methylated base sequence It may have a sequence.
본 발명의 일 예에 따른 ΔTm은 하기의 식 (1)을 만족하는 것을 특징으로 한다.ΔTm according to an embodiment of the present invention is characterized in that it satisfies Equation (1) below.
ΔTm=kXmCXn 식 (1)ΔTm=kXmCXn Equation (1)
[상기 식에서 k는 상수, mC는 메틸화된 시토신이 1개일 경우의 Tm의 차이, n은 메틸화된 시토신의 개수이다.][In the above formula, k is a constant, mC is the difference in Tm when there is one methylated cytosine, and n is the number of methylated cytosines.]
상기 ΔTm은 메틸화된 시토신이 1개일 경우 2℃ 이상 5℃ 이하로 나타날 수 있다.The ΔTm may be 2°C or higher and 5°C or lower when one methylated cytosine is used.
본 발명의 일 실시예에 따른 상기 검출 방법은 표준 PCR, 실시간 PCR(Real Time PCR), 디지털 PCR, 등온PCR(Isothermal PCR), 정량 PCR, DNA 칩, DNA FISH (Fluorescence in situ hybridization)에서 선택되는 어느 하나일 수 있다. The detection method according to an embodiment of the present invention is standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), quantitative PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization) selected from It can be any one.
상기 검출 방법은 유전자 증폭 효율 또는 PCR 산물을 검출할 수 있는 방법을 포함하는 것일 수 있다. 바람직하게는 실시간 PCR을 사용하는 것일 수 있으나 반드시 이에 한정되는 것은 아니며, 상기 PCR은 이 기술 분야에서 사용되는 것이라면 제한 없이 사용될 수 있다. The detection method may include a method capable of detecting gene amplification efficiency or a PCR product. Preferably, real-time PCR may be used, but it is not necessarily limited thereto, and the PCR may be used without limitation as long as it is used in the art.
상기 프로브는 예를 들어, 리덕션 프로브일 수 있다. 상기 리덕션 프로브(저해 프로브; reduction probe)는 메틸화된 검출 부위에 결합하여 유전자의 증폭을 저해하는 프로브일 수 있다. 비메틸화된 검출 부위보다 메틸화된 검출 부위에 더욱 강하게 결합하여 증폭의 차이를 유도할 수 있다.The probe may be, for example, a reduction probe. The reduction probe (inhibition probe; reduction probe) may be a probe that binds to a methylated detection site and inhibits gene amplification. It is possible to induce a difference in amplification by binding more strongly to the methylated detection site than to the unmethylated detection site.
메틸화가 일어날 수 있는 유전자의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재 하에, 상기 유전자에 대한 PCR을 수행하는 단계에서는 메틸화된 시토신과 리덕션 프로브가 상보적 결합을 이룬다. 상보적 결합은 메틸화된 시토신과 리덕션 프로브의 구아닌 염기가 3개의 수소 결합을 함으로써 강한 분자 간의 인력을 형성하는 것이다.In the step of performing PCR on the gene in the presence of a reduction probe capable of specifically binding to a nucleotide sequence of a gene capable of methylation, the methylated cytosine and the reduction probe form complementary binding. Complementary bonding is to form a strong intermolecular attraction by forming three hydrogen bonds between the methylated cytosine and the guanine base of the reduction probe.
메틸화된 시토신은 피리미딘 고리의 5번째 탄소에 메틸화가 되어 있는 시토신을 가리키는데 메틸기는 전자 주는 기(Electron Donation Group; EDG)에 해당하므로 메틸화된 시토신은 상보적 염기에 해당하는 구아닌과 더 강한 수소 결합을 하게 된다. 구아닌과 메틸화된 시토신의 분자간 상호 작용의 증가는 C5-치환체의 전자 공여 특성의 증가와 관련이 있다. 또한, 5-메틸 시토신을 포함하는 쌍만이 표준 시토신에 의해 형성된 구아닌과 시토신 쌍보다 더 강하게 결합한다. 이 결과 PCR 수행시 메틸화된 DNA와 리덕션 프로브 간의 Tm은 비메틸화된 DNA와 리덕션 프로브 간의 Tm 보다 높은 값을 가진다.Methylated cytosine refers to a cytosine that is methylated at the 5th carbon of the pyrimidine ring. Since the methyl group corresponds to an electron donation group (EDG), the methylated cytosine is a complementary base, guanine, and stronger hydrogen. make a bond An increase in the intermolecular interaction of guanine and methylated cytosine is associated with an increase in the electron-donating properties of the C5-substituent. Also, only pairs containing 5-methyl cytosine bind more strongly than the guanine and cytosine pairs formed by standard cytosines. As a result, when PCR is performed, the Tm between the methylated DNA and the reduction probe has a higher value than the Tm between the unmethylated DNA and the reduction probe.
상기 단계에서 수행된 PCR에서의 ΔTm과 ΔCt값 중 적어도 하나 이상을 측정한다. 메틸화된 시토신을 가진 표적 DNA의 경우 비메틸화된 DNA에 비하여 리덕션 프로브와 더 강한 결합을 함으로써 PCR 수행 시 증폭 억제 효과가 나타난다. 이를 이용하여 메틸화가 일어난 유전자의 Tm값 또는 Ct값이 높게 측정이 되고, 메틸화가 일어난 유전자의 Tm값에서 메틸화가 일어나지 않은 유전자의 Tm값을 뺀 ΔTm의 수치 또는 메틸화가 일어난 DNA의 Ct값에서 메틸화가 일어나지 않은 DNA의 Ct값을 뺀 ΔCt의 수치를 확인함으로써 표적 DNA의 메틸화 여부를 판단할 수 있다.At least one of ΔTm and ΔCt values in the PCR performed in the above step is measured. In the case of target DNA having methylated cytosine, the effect of inhibiting amplification appears during PCR by stronger binding with the reduction probe compared to unmethylated DNA. Using this, the Tm value or Ct value of the methylated gene is measured to be high, and the value of ΔTm obtained by subtracting the Tm value of the gene without methylation from the Tm value of the methylated gene or the methylation value from the Ct value of the methylated DNA Whether or not the target DNA is methylated can be determined by checking the value of ΔCt by subtracting the Ct value of the DNA in which the methylation does not occur.
ΔΔCt(cycle threshold)값을 특정하여 메틸화의 정도(X)를 판단하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법을 제공한다. 상기 ΔΔCt는 (리덕션 프로브의 첨가 없는 유전자 시료의 ΔCt-리덕션 프로브를 첨가한 유전자 시료의 ΔCt)이고, ΔCt는(메틸화되지 않은 유전자의 Ct값-메틸화된 유전자의 Ct값)을 나타낸다.Provided is a method for detecting methylation of a gene using a reduction probe that determines the degree (X) of methylation by specifying a ΔΔCt (cycle threshold) value. The ΔΔCt is (ΔCt of the gene sample without the addition of the reduction probe-ΔCt of the gene sample with the addition of the reduction probe), and ΔCt is (Ct value of unmethylated gene - Ct value of methylated gene).
증폭이 저해되면 Ct값이 높게 나타나는 결과 메틸화된 DNA의 ΔCt>0 이고, ΔΔCt<0의 값을 가진다. 메틸화 정도는 0 내지 1사이의 값을 나타낼 수 있다. 1에 가까울수록 메틸화 정도가 낮으며 0에 가까울수록 메틸화 정도가 높은 것을 의미한다. When amplification is inhibited, the Ct value is high. As a result, the methylated DNA has a value of ΔCt>0 and ΔΔCt<0. The degree of methylation may represent a value between 0 and 1. The closer to 1, the lower the methylation degree, and the closer to 0, the higher the methylation degree.
상기 유전자 증폭에 사용되는 프라이머 및 프로브는 서열번호 1 내지 서열번호 30으로 구성된 군에서 선택되는 하나 이상을 포함할 수 있다. 본 발명은 또한, 서열번호 1 내지 서열번호 30으로 구성된 군에서 선택되는 하나 이상을 포함하는, 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브에 관한 것이다. The primers and probes used for the gene amplification may include one or more selected from the group consisting of SEQ ID NOs: 1 to 30. The present invention also relates to a primer or probe for detecting a methylated lung cancer target gene, comprising at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
본 발명은 또한, 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브를 포함하는 폐암 조기 진단용 조성물에 관한 것이다. The present invention also relates to a composition for early diagnosis of lung cancer comprising a primer or probe for detecting a methylated lung cancer target gene.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물을 검체에 처리하는 단계를 포함하는 폐암 조기 진단방법에 관한 것이다.The present invention further relates to a method for early diagnosis of lung cancer comprising treating a sample with the primer or probe for detecting the methylated lung cancer target gene, or the composition.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물의 진단 용도에 관한 것이다. The present invention further relates to a diagnostic use of the primer or probe for detecting the methylated lung cancer target gene, or the composition.
본 발명은 더욱이, 상기 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 상기 조성물을 포함하는 폐암 조기 진단용 키트에 관한 것이다. The present invention further relates to a kit for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene, or the composition.
상기 키트는 샘플을 담는 구획된 캐리어 수단, 주형 DNA를 포함할 수 있는 첫번째 용기, 샘플을 증폭하기 위한 5'-CpG-3' 염기서열 부위에 상보적인 프라이머를 함유하는 두번째 용기, 및 증폭된 산물을 검출하기 위한 프로브를 포함하는 세번째 용기를 포함하는 하나 이상의 용기를 포함한다.The kit comprises a compartmentalized carrier means for holding a sample, a first container capable of containing template DNA, a second container containing a primer complementary to a 5'-CpG-3' sequence region for amplifying the sample, and an amplified product one or more vessels comprising a third vessel containing a probe for detecting
캐리어 수단은 병, 튜브와 같은 하나 이상의 용기를 함유하기에 적합하고, 각 용기는 본 발명의 방법에 사용되는 독립적 구성요소들을 함유한다. 본 발명의 명세서에서, 당해 분야의 통상의 지식을 가진 자는 용기 중의 필요한 제제를 손쉽게 분배할 수 있다.The carrier means is suitable for containing one or more containers, such as bottles and tubes, each container containing the independent components used in the method of the present invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in a container.
실시예 1: 페암 조기진단용 유전자 메틸화 마커 선별Example 1: Selection of gene methylation markers for early diagnosis of lung cancer
선행연구 및 오픈소스 데이타를 바탕으로 정상인과 폐암 환자군에서 발현되는 유전자 정보를 확보하였으며, 정상 대비 암환자에서 메틸화 비율이 80% 이상인 유전자를 선별하였다. 이중 실험적 유의미한 결과를 가지는 유전자 5종을 선택하였다. Genetic information expressed in normal individuals and lung cancer patients was obtained based on previous studies and open source data, and genes with a methylation ratio of 80% or more in cancer patients compared to normal were selected. Among them, five genes with significant experimental results were selected.
실시예 2: 유전자 메틸화 마커 검출을 위한 프라이머/프로브 최적화.Example 2: Primer/Probe Optimization for Gene Methylation Marker Detection.
선별된 5종의 유전자의 메틸화 여부 확인을 위한 PCR 증폭용 프라이머, 프로브를 제작하였으며, 최적화를 통해 최종 아래와 같이 선택하였다. Primers and probes for PCR amplification were prepared to check the methylation of the five selected genes, and the final selection was made as follows through optimization.
실시예 3: 표준물질 농도에 따른 최소검출한계Example 3: Minimum detection limit according to standard concentration
메틸화 판단 표준물질은 Human HCT116 DKO Methylated DNA를 사용하였으며, 검출농도는 0.5, 1, 2.5, 5, 10, 15 ng/ul 이며, 각 농도당 5회 반복을 진행하였다. 최소검출한계 판단기준은 변동계수(%) 5 미만으로 설정하였으며, 농도에 따른 최소검출 한계는 10ng으로 확인되었다. Human HCT116 DKO Methylated DNA was used as the standard for methylation determination, and the detection concentrations were 0.5, 1, 2.5, 5, 10, and 15 ng/ul, and each concentration was repeated 5 times. The minimum detection limit judgment criterion was set to a coefficient of variation (%) less than 5, and the minimum detection limit according to the concentration was confirmed to be 10ng.
실시예 4: 표준물질 메틸화 비율(%)에 따른 최소검출한계Example 4: Minimum detection limit according to standard methylation ratio (%)
메틸화 판단 표준물질은 Human HCT116 DKO Methylated DNA를 사용하였으며, 메틸화 비율은 0.5, 1, 5, 10, 50, 100% 이며, 각 농도당 5회 반복을 진행하였다. 최소검출한계 판단기준은 변동계수(%) 5 미만으로 설정하였으며, 메틸화 비율에 따른 최소검출 한계는 1% 로 확인되었다. Human HCT116 DKO Methylated DNA was used as a standard for determining methylation, and methylation rates were 0.5, 1, 5, 10, 50, and 100%, and each concentration was repeated 5 times. The minimum detection limit judgment criterion was set to a coefficient of variation (%) less than 5, and the minimum detection limit according to the methylation ratio was confirmed to be 1%.
실시예 5: 표준물질을 사용한 재현성 비교Example 5: Comparison of reproducibility using standards
메틸화 판단 표준물질은 Human HCT116 DKO Methylated DNA를 사용하였으며, 3개의 lot, 3대의 장비, 3명의 시험자가 각 실험별 3반복씩 수행하여 총 81회 반복 실험을 진행하였다. 재현성 판단기준은 변동계수(%) 5 미만으로 설정하였으며, 그 결과 CV 5 미만으로 확인되었다. Human HCT116 DKO Methylated DNA was used as the standard for methylation determination, and 3 lots, 3 equipment, and 3 testers performed 3 repetitions for each experiment, a total of 81 repetitions were performed. The reproducibility criterion was set to a coefficient of variation (%) less than 5, and as a result, it was confirmed to be less than CV 5.
실시예 6: 기존 메틸화 분석법 (pyrosequncing)과 정확도 비교Example 6: Comparison of accuracy with conventional methylation assay (pyrosequuncing)
각 샘플은 3회 반복을 진행하여 평균을 구했으며, 빈도는 0 ~ 100% 구간을 실험하였다. 메틸화 비율(MPR)은 메틸화 바이오 마커 (Target)의 Ct 값에서 하우스키핑유전자 (ACTB)의 Ct 값을 기반으로 산출하였다. 산출식은 아래와 표 4와 같다. Each sample was repeated 3 times to obtain an average, and the frequency ranged from 0 to 100%. The methylation ratio (MPR) was calculated based on the Ct value of the housekeeping gene (ACTB) from the Ct value of the methylation biomarker (Target). The calculation formula is shown in Table 4 below.
시험결과는 아래 표와 같으며, 본 발명의 메틸화 분석법이 기존 대비 정확도가 높은 것을 확인하였다. The test results are shown in the table below, and it was confirmed that the methylation analysis method of the present invention has higher accuracy than the existing ones.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. 본 발명의 단순한 변형 내지 변경은 이 분야의 통상의 지식을 가진 자에 의하여 용이하게 이용될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.
본 발명에 따르면, 폐암 조기진단을 위한 저선량 흉부 CT (LDCT)의 한계를 극복하고, 보다 정확한 유전자 메틸화 여부 분석 방법 및 이를 위한 키트 제공은 건강검진 및 폐암 조기진단에 효과적이다. According to the present invention, it is effective to overcome the limitations of low-dose chest CT (LDCT) for early diagnosis of lung cancer, and to provide a more accurate method for analyzing whether gene methylation is present and a kit for the same, for medical examination and early diagnosis of lung cancer.
Claims (9)
- 메틸화된 폐암 표적 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 표적 유전자를 증폭시키는 단계; amplifying the target gene in the presence of a PNA probe capable of hybridizing with the methylated lung cancer target gene;비메틸화 유전자와 혼성화할 수 있는 PNA 프로브의 존재하에 메틸화되지 않은 유전자를 증폭시키는 단계; 및amplifying the unmethylated gene in the presence of a PNA probe capable of hybridizing with the unmethylated gene; and상기 증폭 산물의 융해곡선을 분석하여 표적 유전자의 메틸화 여부를 판별하는 단계를 포함하는, 폐암 조기진단을 위한 유전자의 메틸화 검출 방법.A method for detecting methylation of a gene for early diagnosis of lung cancer, comprising the step of analyzing a melting curve of the amplification product to determine whether a target gene is methylated.
- 제1항에 있어서, 상기 증폭은 표준 PCR, 실시간 PCR(Real Time PCR), 디지털 PCR, 등온 PCR(Isothermal PCR), DNA 칩, DNA FISH (Fluorescence in situ hybridization)에서 선택되는 어느 하나에 의해 수행되는 것을 특징으로 하는 유전자의 메틸화 검출 방법.The method of claim 1, wherein the amplification is performed by any one selected from standard PCR, real time PCR, digital PCR, isothermal PCR, DNA chip, and DNA FISH (Fluorescence in situ hybridization). A method for detecting methylation of a gene, characterized in that.
- 제1항에 있어서, 상기 메틸화 검출 방법은 유전자의 증폭 없이, 메틸화된 또는 비메틸화 유전자와 프로브의 ΔTm (melting temperature) 및/또는 ΔCt(cycle threshold) 값을 측정하여 메틸화 여부 또는 메틸화 정도를 분석하는 단계를 포함하는, 유전자의 메틸화 검출 방법.According to claim 1, wherein the methylation detection method is to analyze whether methylation or the degree of methylation by measuring ΔTm (melting temperature) and/or ΔCt (cycle threshold) values of a methylated or unmethylated gene and a probe without amplification of the gene A method for detecting methylation of a gene, comprising the step.
- 제1항에 있어서, 상기 검출 방법은 The method of claim 1, wherein the detection methoda. 메틸화된 표적 유전자의 염기서열과 특이적으로 결합할 수 있는 PNA 프로브의 존재하에, 표적 유전자에 대한 PCR을 수행하는 단계; a. performing PCR on the target gene in the presence of a PNA probe capable of specifically binding to the methylated base sequence of the target gene;b. 비메틸화 유전자의 염기서열과 특이적으로 결합할 수 있는 PNA 프로브의 존재하에, 상기 메틸화 되지 않은 유전자에 대한 PCR을 수행하는 단계; b. performing PCR on the unmethylated gene in the presence of a PNA probe capable of specifically binding to the base sequence of the unmethylated gene;c. 상기 PCR에서의 ΔTm (melting temperature) 및 ΔCt (cyclethreshold) 값 중 하나 이상을 측정하는 단계; c. measuring one or more of ΔTm (melting temperature) and ΔCt (cyclethreshold) values in the PCR;d. ΔTm 또는 ΔCt의 수치를 통해 유전자의 메틸화 여부를 판단하거나, 또는 ΔΔCt값을 특정하여 메틸화 비율(MPR)를 판단하는 단계를 포함하는 것을 특징으로 하는 유전자의 메틸화 검출 방법.d. A method for detecting methylation of a gene, comprising the step of determining whether a gene is methylated through a value of ΔTm or ΔCt, or determining a methylation ratio (MPR) by specifying a ΔΔCt value.
- 제1항에 있어서, 상기 표적 유전자의 염기서열은 메틸화된 염기서열, 하이드록시 메틸화된 염기서열, 포르밀 메틸화된 염기서열, 카르복실 메틸화된 염기서열로 구성된 군에서 선택되는 하나 이상을 포함하는, 유전자의 메틸화 검출 방법.According to claim 1, wherein the base sequence of the target gene comprises at least one selected from the group consisting of a methylated base sequence, a hydroxymethylated base sequence, a formyl methylated base sequence, and a carboxyl methylated base sequence, A method for detecting methylation of a gene.
- 제1항에 있어서, 상기 유전자 증폭에 사용되는 프라이머 및 프로브는 서열번호 1 내지 서열번호 30으로 구성된 군에서 선택되는 하나 이상을 포함하는, 유전자의 메틸화 검출 방법.The method of claim 1, wherein the primers and probes used for gene amplification include at least one selected from the group consisting of SEQ ID NOs: 1 to 30.
- 서열번호 1 내지 서열번호 30으로 구성된 군에서 선택되는 하나 이상을 포함하는, 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브.A primer or probe for detecting a methylated lung cancer target gene, comprising at least one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
- 제7항의 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브를 포함하는 폐암 조기 진단용 조성물.A composition for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene of claim 7.
- 제7항의 메틸화된 폐암 표적 유전자 검출용 프라이머 또는 프로브, 또는 제8항의 조성물을 포함하는 폐암 조기 진단용 키트.A kit for early diagnosis of lung cancer comprising the primer or probe for detecting the methylated lung cancer target gene of claim 7, or the composition of claim 8.
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