CN111118162A - JAK2 deletion mutant gene and detection method thereof - Google Patents
JAK2 deletion mutant gene and detection method thereof Download PDFInfo
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Abstract
The invention provides a specific human JAK2 mutant gene for the first time, the mutant gene is formed by c. [ 84709-: 2, respectively. The colorectal cancer patient with the JAK2 gene c [84709-84714delctgcag ] mutation has high risk of lung metastasis, and the marker can be used as an effective and reliable molecular marker for the lung metastasis of colorectal cancer; the invention also provides a JAK2 mutant gene detection and analysis method based on PCR amplification and Sanger sequencing, which can be simply, conveniently and effectively applied to analysis of lung metastasis of colorectal cancer patients, is beneficial to early discovery and early intervention of lung metastasis of colorectal cancer patients, and can also provide clues for mechanism analysis of colorectal cancer lung metastasis.
Description
Technical Field
The invention relates to the field of medical molecular biology, in particular to a JAK2 mutant gene with deletion mutation at the 84709-84714 site c. [84709-84714delctgcag ], and an application thereof.
Background
Colorectal cancer is one of the high-incidence cancer species of Chinese people, has high malignancy degree, is easy to generate metastasis and has high lethality rate. According to the latest release of the annual report of cancers in 2018 by WHO, the incidence and mortality of colorectal cancer are high in the 4 th of common cancers of Chinese people. Clinically, combined treatment mainly comprising surgery and assisted chemoradiotherapy is mostly adopted for colorectal cancer, but the effect is not ideal, and the cause of death is mainly metastasis and recurrence after radical surgery. Currently, among metastases of colorectal cancer patients, the lung is a very common target organ for blood circulation metastasis, which has become the second most common metastatic site next to the liver. Retrospective data from 1996 to 2017 in the tumor hospital, Beijing university, show that lung metastasis cases account for 32.9% of all metastatic colorectal cancers, while patients with incipient lung metastasis reach 24.5%. Among patients with primary pulmonary metastasis, simple pulmonary metastasis accounts for 37.7% -44.5%, and only 21.1% -32.5% of patients can receive radical resection operation treatment of pulmonary metastasis.
The early discovery of the lung metastasis of the colorectal cancer is very critical, early prevention and treatment can be realized based on the early discovery, and the early discovery mainly can increase the chance of radical resection of the lung metastasis of a patient and carry out preventive treatment in a targeted manner. Because the molecular mechanism of the colorectal cancer lung metastasis is not analyzed, an accurate and reliable colorectal cancer lung metastasis prediction or evaluation index is not available clinically at present, and sensitivity and specificity of some clinical characteristics and serological indexes are insufficient, so that the lung metastasis risk of a colorectal cancer patient cannot be analyzed effectively and substantially as early as possible. Cancer is essentially a genetic disease whose development is driven by the variation of a specific gene group, with specific genetic indications. Therefore, the mechanism of the colorectal cancer lung metastasis can be revealed only by exploring the gene level, and corresponding molecular markers can be found out, so that the lung metastasis of the colorectal cancer patient can be predicted or discovered as soon as possible, which has important significance and value for timely taking necessary clinical intervention or treatment measures to practically improve the survival rate of the colorectal cancer patient.
According to the invention, a specific colorectal cancer family in Fujian province is firstly collected, the whole exome sequencing is carried out on related family members, two specific deletion mutations of JAK2 gene, namely c. [41905 and 41909deltctga ] and c. [84709 and 84714delctgcag ] are found in colorectal cancer lung metastasis patients for the first time through analysis and comparison, and the verification is further carried out on the colorectal cancer patient population through the PCR amplification and Sanger sequencing of mutation fragments. JAK2(Janus kinase 2) kinase coded by JAK2 gene is an important member in protein tyrosine kinase family JAKs, and JAKs is an important signal sensor of a plurality of cytokines, growth factors and interferons, relates to a plurality of important signal channels (such as JAK/STAT channels and the like) from a membrane to a nucleus, is widely involved in the processes of proliferation, differentiation, apoptosis, immune regulation and the like of cells, and specific mutation of the JAK/STAT signal channels can cause cell runaway. The JAK2 deletion mutant gene provided by the invention has not been reported, and the clinical significance and value of the JAK2 deletion mutant gene to lung metastasis of colorectal cancer patients have not been disclosed.
Disclosure of Invention
The invention provides a specific human JAK2 deletion mutant gene for the first time, which can be used as a molecular marker of colorectal cancer lung metastasis, and provides a JAK2 deletion mutant gene detection analysis method based on PCR amplification and Sanger sequencing, which can be applied to analysis of the colorectal cancer patient lung metastasis and can also provide clues for mechanism analysis of the colorectal cancer lung metastasis.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the object (1) of the present invention is to provide a human JAK2 mutant gene, wherein the JAK2 mutant gene is formed by c. [41905 & 41909deltctga ] deletion mutation of the wild-type JAK2 gene, i.e., 5 nucleotide short fragment of tctga at 41905 & 41909 site of the wild-type JAK2 gene is deleted, with reference to the sequence of the wild-type JAK2 gene with NCBI accession number NG _ 009904.1. The sequence of 50bp (containing 100 bp) before and after the mutation position of the JAK2 mutant gene, namely the sequence of the 41855-41954 site of the JAK2 mutant gene is shown as SEQ ID NO: 1, and the rest sequences are detailed in a reference sequence of a wild-type JAK2 gene with NCBI accession number NG _009904.1, and specifically: the sequence of the former 41904 site of the JAK2 mutant gene is the same as the sequence of the same site of a wild type JAK2 gene (NCBI accession number is NG _ 009904.1); the sequence of the JAK2 mutant gene is identical to that of 41909+ N site of the wild-type JAK2 gene from 41904+ N site (N is an integer between 1 and 108035 in the range of 1-108035). The JAK2 gene has c. [41905 and 41909deltctga ] mutation, and the lung metastasis risk of the colorectal cancer patient is high, so the mutant gene can be used as a molecular marker of the lung metastasis of the colorectal cancer patient and applied to the construction of a detection kit and a detection method for the lung metastasis analysis of the colorectal cancer patient.
The object (2) of the present invention is to provide a human JAK2 mutant gene, wherein the JAK2 mutant gene is formed by c. [ 84709-. The sequence of 50bp before and after the mutation position of the JAK2 mutant gene, namely the sequence of the 84659-84758 th site (containing 100bp of mutation) of the JAK2 mutant gene is shown as SEQ ID NO: 2, the remaining sequences are detailed in the reference sequence of the wild-type JAK2 gene with NCBI accession number NG _009904.1, specifically: the sequence of the front 84708 site of the JAK2 mutant gene is the same as the sequence of the same site of a wild-type JAK2 gene (NCBI accession number is NG _ 009904.1); the sequence of the JAK2 mutant gene is identical to that of 84714+ N site of the wild-type JAK2 gene from 84708+ N site (N is an integer between 1 and 65231). The JAK2 gene has c. [84709-84714delctgcag ] mutation, and the colorectal cancer patient has high risk of lung metastasis, so the mutant gene can be used as a molecular marker of the lung metastasis of the colorectal cancer patient and applied to the construction of a detection kit and a detection method for the lung metastasis analysis of the colorectal cancer patient.
The object (3) of the present invention is to provide a method for detecting and analyzing a specific JAK2 mutant gene in a colorectal cancer patient, which comprises the following main steps:
(1) extraction of DNA from colorectal cancer patient samples
Obtaining a cancer focus tissue or a blood sample of a colorectal cancer patient, wherein the cancer focus tissue can be a sample obtained by operation or biopsy puncture, the blood sample can be peripheral venous blood convenient to obtain, and extracting genome DNA in the cancer focus tissue sample or extracellular free DNA in the blood sample by selecting a corresponding kit.
(2) Amplification of fragment in which mutation of JAK2 gene is located
Using the DNA extracted in the step (1) as a template, and adopting the DNA shown in SEQ ID NO: 3 and SEQ ID NO: 4, amplifying by using the PCR primer shown in the sequence according to a conventional PCR amplification system under the following amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 20s, and extension at 72 ℃ for 40s, and circulating for 30 times; final extension at 72 deg.C for 5 min;
for the c. [ 84709-: 5 and SEQ ID NO: 6, amplifying by using a PCR primer shown in a sequence according to a conventional PCR amplification system under the following amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 35s, and circulating for 30 times; final extension at 72 deg.C for 5 min;
(3) sequencing analysis of JAK2 mutant gene amplification fragment
The JAK2 gene mutation fragment amplified in the step (2) is sent to a gene company for Sanger sequencing, and specific mutation information of a JAK2 gene in a sample is analyzed by referring to a reference sequence of a wild type JAK2 gene with the NCBI accession number of NG _009904.1, so that the JAK2 gene mutation fragment is applied to analysis of lung metastasis of a colorectal cancer patient. When a c. [ 41905. sup. 41909. deltctga ] mutation in JAK2 gene and/or a c. [ 84709. sup. 84714. delctgcag ] mutation in JAK2 gene were detected in a sample of a patient with colorectal cancer, it was indicated that the mutation was a lung metastasis or a high risk of lung metastasis.
The human JAK2 mutant gene provided by the invention can be used as an effective and reliable molecular marker for colorectal cancer lung metastasis, and can also provide clues for mechanism analysis of colorectal cancer lung metastasis; the JAK2 mutant gene and the detection method thereof can be simply, conveniently and effectively applied to the analysis and prediction of the lung metastasis of the colorectal cancer patient, and are beneficial to the early discovery and early intervention of the lung metastasis of the colorectal cancer patient.
Drawings
FIG. 1 is a partial map of Sanger sequencing before and after the c. [41905-41909deltctga ] mutation for the wild-type gene (A) and the mutant gene (B) of JAK 2. The boxed region in the figure is the region where the deletion mutation occurs.
FIG. 2 is a partial map of Sanger sequencing before and after the c. [ 84709-. The boxed region in the figure is the region where the deletion mutation occurs.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1 Presence of a mutation in the JAK2 specific Gene in patients with colorectal cancer pulmonary metastasis
Specific colorectal cancer families in Fujian province are collected, each family contains 2 clinically and pathologically diagnosed colorectal cancer patients who are subjected to resection (sibling siblings are among patients in the same family, and the diagnosis time interval is counted to be 3-32 months), wherein the specific information of the patients of 10 families containing at least 1 lung metastasis is shown in Table 1. When researching molecular indications of colorectal cancer metastasis, the same family with a plurality of colorectal cancer patients is preferably selected, so that interference of the genetic background of the population can be eliminated, and the same and/or different mutation information can be found out more easily. Theoretically, different colorectal cancer patients in the same family sibling have higher cancer focus homology, the same molecular indication should be obtained if the colorectal cancer patients are transferred to the same visceral organ, different molecular indications should be obtained if the colorectal cancer patients are transferred to different visceral organs, and genetic background interference can be eliminated by selecting the same family member, so that the molecular indications can be conveniently found. In addition, the selection of sibling colorectal cancer patients in the same family facilitates the collection of analytical samples in the case where these members are all alive.
Postoperative tissue samples were taken with adequate informed consent and satisfaction of clinical diagnosis in table 1 for 10 family-related colorectal cancer patients. The genomic DNA of the Tissue sample of the patient to be tested was extracted using a Tissue genome extraction Kit (QIAamp Fast DNA Tissue Kit and QIAamp DNA FFPE Tissue Kit) from Qiagen, Germany, according to the method described in the specification. And after the genome DNA is detected to be qualified, the genome DNA is sent to Beirui and congong department for whole exome capture sequencing and analysis. The capture sequencing library is constructed by adopting a SureSelect Human All Exon V6+ UTR kit of Agilent, and sequencing is carried out by adopting a HiSeq2500 sequencer of Illumina, and the average sequencing depth reaches 220 x. Performing quality control and standard pre-treatment such as removing low-quality reads on sequencing original data, taking hg19 genome as reference, and identifying and annotating variable site information such as SNV, Indel, CNV and the like by using BWA, GATK and other software; and finally, determining mutation site information by using invalid filtering and polymorphism information of databases such as a dbSNP database, a thousand-people genome database and an exome sequencing project database ESP6500, and sorting important mutations by combining clinical information and then performing Sanger sequencing verification.
The mutation status of the JAK2 gene for 10 families of colorectal cancers is shown in Table 1. The results showed that in 16 colorectal cancer lung metastasis patients in 10 selected families, except 2 lung metastasis patients in family #9, no JAK2 gene mutation was detected, and in all the 14 lung metastasis patients, a specific mutation of JAK2 gene, namely, c. [ 41905. sup. 41909. deltctga ] mutation or c. [ 84709. sup. 84714. delctgcag ] mutation was detected, and the content ratio of the mutations (the percentage of the number of reads of the specific mutation of JAK2 gene contained in the sequencing effective mutation reads) was more than 3% (3.6% -11.1%). Wherein 8 patients with colorectal cancer lung metastasis contained in patients 1 and 2 at family #1, patient 1 at family #2, patient 2 at family #3, patient 2 at family #5, patient 1 at family #6, patient 2 at family #7 and patient 2 at family #10 are all the c. [41905 and 41909deltctga ] mutations of JAK2 gene, and 6 patients with colorectal cancer lung metastasis contained in patients 2 at family #2, patient 1 at family #3, patients 1 and 2 at family #4, patient 1 at family #5 and patient 1 at family #8 are all the c. [84709 and 84714delctgcag ] mutations of JAK2 gene. It was thus shown that specific mutations of JAK2 gene were prevalent in colorectal cancer lung metastasis patients (14 cases/16 cases = 87.5%), whereas individual patients without specific mutations of JAK2 gene (2 patients of family # 9) were presumed to be sporadic and further need to be validated in a large population of colorectal cancer patients.
TABLE 110 family information on colorectal cancer and JAK2 Gene mutation status
| Family number | Patient 1 | Mutations and their ratios | Patient 2 | Mutations and their ratios |
| #1 | For male, age 63, lung metastasis | c.[41905-41909deltctga]，8.4% | For male, age 65, lung metastasis | c.[41905-41909deltctga]，10.3% |
| #2 | Female, age 54, lung metastasis | c.[41905-41909deltctga]，7.0% | Male, age 55, lung metastasis | c.[84709-84714delctgcag]，6.2% |
| #3 | Female, age 49, lung metastasis | c.[84709-84714delctgcag]，5.7% | Male, age 51, lung metastasis | c.[41905-41909deltctga]，11.1% |
| #4 | Female, age 63, with lung metastasis | c.[84709-84714delctgcag]，8.5% | Female, age 64, lung metastasis | c.[84709-84714delctgcag]，4.9% |
| #5 | Female, age 64, lung metastasis | c.[84709-84714delctgcag]，3.6% | Female, age 65, lung metastasis | c.[41905-41909deltctga]，7.7% |
| #6 | Female, age 63, with lung metastasis | c.[41905-41909deltctga]，8.3% | For male, age 64, liver metastasis | - |
| #7 | A male, age 60, liver metastasis | For male, age 65, lung metastasis | c.[41905-41909deltctga]，6.5% | |
| #8 | For male, age 46, lung metastasis | c.[84709-84714delctgcag]，4.8% | For male, age 48, liver metastasis | - |
| #9 | Male, age 51, lung metastasis | - | For male, age 54, lung metastasis | - |
| #10 | A male, age 60, liver metastasis | - | For male, age 62, lung metastasis | c.[41905-41909deltctga]，6.7% |
Note: "-" indicates no detection.
Example 2 exploration of JAK2 specific Gene mutations in a population of patients with rectal cancer
In order to further explore the distribution rule of JAK2 specific gene mutations, namely c. [ 41905. sup. 41909deltctga ] and c. [ 84709. sup. 84714delctgcag ] in the rectal cancer patient population, PCR primers for the two specific mutations were designed and 127 postoperative tissue samples of colorectal cancer patients diagnosed with clinical pathology in Fujian province were collected, a sample genomic DNA was extracted according to the method in example 1, the extracted genomic DNA was used as a template, fragments in which mutations were amplified according to the conventional PCR system and the conditions in Table 2 (each sample was amplified by using the primer pair of Seq ID NO: 3 and Seq ID NO: 4 and the primer pair of Seq ID NO: 5 and Seq ID NO: 6 separately), and the PCR products were sent to Sesheng Bioengineering Co Ltd for Sanger sequencing after electrophoresis detection. The results of Sanger sequencing of JAK2 specific gene mutation in 127 colorectal cancer patients are shown in Table 3, and the results show that 32 colorectal cancer lung metastasis patients all detected specific gene mutation containing JAK2, namely c. [41905 + 41909 deltcga ] (19 cases, Sanger sequencing map at mutation site is shown in FIG. 1) or c. [84709 + 84714delctgcag ] (13 cases, Sanger sequencing map at mutation site is shown in FIG. 2), while non-lung metastasis patients all did not detect specific mutation of JAK2 gene.
TABLE 2 mutant fragment amplification systems and conditions
| Type of mutation | Amplification primers | Amplification conditions |
| c.[41905-41909deltctga] | Seq ID NO: 3 and Seq ID NO: 4 | Pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 52 ℃ for 20s, extension at 72 ℃ for 30s, and circulation for 30 times; final extension at 72 ℃ for 5 min. |
| c.[84709-84714delctgcag] | Seq ID NO: 5 and Seq ID NO: 6 | Pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 54 ℃ for 20s, and extension at 72 ℃ for 40s, and circulating for 30 times; final extension at 72 ℃ for 5 min. |
TABLE 3 mutation profiles of JAK2 specific genes in colorectal cancer patient populations
| Colorectal cancer patient type | Total number of | c.[41905-41909deltctga] | c.[84709-84714delctgcag] |
| Pulmonary metastasis in males | 15 | 10 | 5 |
| Non-pulmonary metastasis in males | 52 | 0 | 0 |
| Female with a view to preventing the formation of wrinklesMetastasis of lung | 17 | 9 | 8 |
| Non-pulmonary metastasis in females | 43 | 0 | 0 |
| Summary statistics | 127 | 19 | 13 |
Further, 8 lung metastasis patients and 8 non-lung metastasis patients are selected from 127 colorectal cancer patients, and PCR capture sequencing of JAK2 whole genes is carried out by a primer set designed by a gene delivery company, and the result shows that the non-lung metastasis patients do not have specific mutations of two JAK2 genes, and also do not have other mutations of JAK2 genes at any other sites and types; the JAK2 gene mutation of lung metastasis patients is mainly two types, namely c [41905 & 41909deltctga ] and c [84709 & 84714delctgcag ]. Thus, patients with colorectal lung metastasis have one of two specific mutations of JAK2 gene, namely c. [41905 & 41909deltctga ] or c. [84709 & 84714delctgcag ]. Therefore, the two specific mutations of the JAK2 gene can be used as molecular markers of lung metastasis of a colorectal cancer patient, and whether the JAK2 gene has the capability of predicting the lung metastasis of the colorectal cancer patient needs to be further researched.
Example 3 mutation of JAK2 specific genes can predict lung metastasis in colorectal cancer patients
Collecting 50 clinically and pathologically diagnosed colorectal cancer patients (both feasible resection and impossible resection) in Fujian province, wherein the colorectal cancer patients need to meet the first diagnosis of colorectal cancer and have no metastasis and no other tumors in the initial diagnosis, collecting 2ml of peripheral venous blood before hospitalization, and additionally extracting 2ml of peripheral venous blood every 3 months when blood sampling is needed for examination and treatment until the patients have clinical images and pathologically diagnosed metastasis. There were 42 final patients who were effective, and 8 additional patients who had died or missed the study. The free nucleic acid was extracted from all blood samples of the 42 patients who were active using the Qiagen extracellular free nucleic acid extraction Kit (Circulating nucleic acid Kit). Two specific mutant fragments of JAK2 gene were amplified using the extracted blood free nucleic acid as a template according to the system and conditions of table 2 in example 2, and after electrophoretic detection, PCR products were sent to shanghai bio-engineering ltd for Sanger sequencing, with the results shown in table 4.
The results showed that 12 of the metastatic foci after 42 colorectal cancer patients were lungs, 23 were non-lungs (including liver, bone and brain), and 7 did not develop metastasis, wherein two specific mutations of JAK2 gene were not detected in 23 non-lung metastatic patients and 7 non-metastatic patients. The 12 patients with lung metastases all contained one of two specific mutations in the JAK2 gene, 7 of them contained the c. [ 41905. sup. 41909. deltctga ] mutation in the JAK2 gene, and 5 contained the c. [ 84709. sup. 84714. delctgcag ] mutation in the JAK2 gene. The 12 colorectal cancer patients all detected specific mutation of JAK2 gene before clinical imaging and/or pathological finding and diagnosis of lung metastasis. Compared with the clinical imaging and/or pathological lung metastasis discovery and diagnosis results, the molecular mutation detection result is averagely 4.7 months earlier (within the range of 3 months to 6 months) for the colorectal cancer lung metastasis patients with the c. [41905 and 41909deltctga ] mutation of the JAK2 gene, and is averagely 7.3 months earlier (within the range of 6 months to 9 months) for the patients with the c. [84709 and 84714delctgcag ] mutation of the JAK2 gene. It was further shown that the c. [ 41905. sup. 41909. deltctga ] and c. [ 84709. sup. 84714. delctgcag ] mutations of JAK2 gene can be used as molecular markers for lung metastasis in patients with colorectal cancer, and patients with colorectal cancer who have detected these two specific gene mutations of JAK2 gene in blood are at high risk of lung metastasis (7 cases/7 cases =100%, 5 cases/5 cases = 100%). Molecular detection would make it more advantageous for the prediction, early detection of metastasis, due to earlier imaging and pathology results, especially in combination with liquid biopsy and second generation genomic sequencing technologies.
Table 442 colorectal cancer patients with metastasis and JAK2 specific gene mutation
| Transfer type | Number of examples | Containing c. [41905-]Number of mutation case Example (b) | Molecular detection mean advance time- Moon cake | Contains c. [84709-]Number of mutation case Example (b) | Molecular detection mean advance time- Moon cake |
| Metastasis of lung | 12 | 7 | 4.7 | 5 | 7.3 |
| Non-pulmonary metastasis | 23 | 0 | - | 0 | - |
| Is not transferred | 7 | 0 | - | 0 | - |
Note: "-" indicates no detection.
Sequence listing
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Claims (3)
1. A JAK2 mutant gene, wherein: with reference to the sequence of the wild-type JAK2 gene with NCBI accession number NG _009904.1, the JAK2 mutant gene is formed by c. [ 84709-; the sequence of the front 84708 site of the JAK2 mutant gene is the same as the sequence of the same site of the wild JAK2 gene; the sequence of the JAK2 mutant gene is the same as the sequence of 84714+ N site of the wild-type JAK2 gene from the 84708+ N site, and N is an integer between 1 and 65231 within the range of 1 to 65231; the sequence of the 84659-84758 th site of the JAK2 mutant gene, namely the sequence of 50bp before and after the mutation is shown as SEQ ID NO: 2 is shown in the specification; the risk of lung metastasis is high in the colorectal cancer patient with the JAK2 gene having c [84709-84714delctgcag ] mutation.
2. The method for the detection and analysis of JAK2 mutant gene according to claim 1, for the detection of JAK2 mutant gene in colorectal cancer patient, wherein: the detection method of the JAK2 mutant gene comprises the following main steps:
(1) extraction of DNA from colorectal cancer patient samples
Obtaining a cancer focus tissue or a blood sample of a colorectal cancer patient, wherein the cancer focus tissue can be a sample obtained by operation or biopsy puncture, the blood sample can be peripheral venous blood convenient to obtain, and genome DNA in the cancer focus tissue sample or extracellular free DNA in the blood sample is extracted by selecting a corresponding kit;
(2) amplification of fragment in which mutation of JAK2 gene is located
Taking the DNA extracted in the step (1) as a template, and adopting the DNA shown in SEQ ID NO: 5 and SEQ ID NO: 6, amplifying by using a PCR primer shown in a sequence according to a conventional PCR amplification system under the following amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 35s, and circulating for 30 times; final extension at 72 deg.C for 5 min;
(3) sequencing analysis of JAK2 mutant gene amplification fragment
The JAK2 gene mutation fragment amplified in the step (2) is sent to a gene company for Sanger sequencing, and specific mutation information of a JAK2 gene in a sample is analyzed by referring to a reference sequence of a wild type JAK2 gene with the NCBI accession number of NG _009904.1, so that the method is applied to analysis of lung metastasis of a colorectal cancer patient: if a c. [84709-84714delctgcag ] mutation containing JAK2 gene is detected in a colorectal cancer patient sample, it indicates that the mutation is lung metastasis or high risk of lung metastasis.
3. The use of the JAK2 mutant gene of claim 1 in constructing a detection kit and a detection method for lung metastasis analysis of colorectal cancer patients.
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| US20160045498A1 (en) * | 2013-04-04 | 2016-02-18 | The Walter And Eliza Hall Institute Of Medical Research | Methods of treating diseases characterized by excessive wnt signalling |
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| US20160045498A1 (en) * | 2013-04-04 | 2016-02-18 | The Walter And Eliza Hall Institute Of Medical Research | Methods of treating diseases characterized by excessive wnt signalling |
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