CN111118163A - JAK1 deletion mutant gene and application thereof - Google Patents
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Abstract
The invention provides a specific human JAK1 mutant gene for the first time, the mutant gene is formed by c. [106282-106290 delcgcgcgcgcgcgcggac ] deletion mutation of a wild type JAK1 gene, and the sequence of the 106232-106331 site, namely the sequence of 50bp before and after the mutation, is shown as SEQ ID NO: 1 is shown. The liver metastasis risk of male colorectal cancer patients with JAK1 gene c. [106282 and 106290delcgccgcgac ] mutation is high, and the male colorectal cancer patients can be used as effective and reliable molecular markers for colorectal cancer liver metastasis; the invention also provides a JAK1 mutant gene detection and analysis method based on PCR amplification and Sanger sequencing, which can be simply, conveniently and effectively applied to analysis of liver metastasis of male colorectal cancer patients, is beneficial to early discovery and early intervention of liver metastasis of male colorectal cancer patients, and can also provide clues for mechanism analysis of liver metastasis of colorectal cancer.
Description
Technical Field
The invention relates to the field of medical molecular biology, in particular to a JAK1 mutant gene with deletion mutation of a 106282-106290 site [106282-106290 delcgccgccgac ] and 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. The liver is the main target organ for colorectal cancer hematogenous metastasis, and colorectal cancer hepatic metastasis is the most important death cause of colorectal cancer patients: 15% -25% of colorectal cancer patients are diagnosed with liver metastasis, and 15% -25% of colorectal cancer patients have liver metastasis after radical treatment of colorectal cancer, wherein most (80% -90%) of liver metastasis foci cannot be radically resected; while the median survival time of untreated colorectal cancer patients with liver metastasis is only 6.9 months, and the 5-year survival rate of patients with liver metastasis unresectable is less than 5%.
Colorectal cancer liver metastasis is the key and difficult point of colorectal cancer treatment, and the key point is how to find liver metastasis as soon as possible to realize early prevention and treatment. Researches find that the 3 factors of lymph node metastasis condition, preoperative carcinoembryonic antigen level and patient onset age have the most obvious influence on whether the colorectal cancer patient has liver metastasis, and have the value of prompting the risk of the liver metastasis. However, these risk factors are either not sensitive enough, not specific enough, or not time-sensitive enough to effectively and accurately predict the risk of liver metastasis in colorectal cancer patients. 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 liver metastasis of colorectal cancer must be revealed by exploring the gene level, and the corresponding molecular marker is found out from the mechanism, so that the liver metastasis of colorectal cancer patients can be predicted or discovered as soon as possible.
According to the invention, specific colorectal cancer families in Fujian province are collected firstly during research, all exome sequencing is carried out on related family members, two specific mutations of JAK1 gene, namely c. [106282 and 106290 delcgccgccgac ] deletion mutation and c. [238094 and 238095instTAT ] insertion mutation, are found in male colorectal cancer liver transfer patients for the first time through analysis and comparison, and further verification is carried out on male colorectal cancer patient population through PCR amplification of mutation fragments and Sanger sequencing. JAK1(Janus kinase 1) kinase coded by JAK1 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 JAK1 mutant gene provided by the invention has not been reported, and the clinical significance and value of the JAK1 mutant gene on liver metastasis of a male colorectal cancer patient have not been disclosed.
Disclosure of Invention
The invention provides a specific human JAK1 mutant gene for the first time, which can be used as a molecular marker of colorectal cancer liver metastasis, and also provides a JAK1 mutant gene detection and analysis method based on PCR amplification and Sanger sequencing, which can be applied to analysis of liver metastasis of male colorectal cancer patients and can also provide clues for mechanism analysis of colorectal cancer liver 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 mutant human JAK1 gene, wherein the JAK1 mutant gene is formed by c. [106282 and 106290 delcgcgcgcggac ] mutation of the wild-type JAK1 gene, i.e., a 9-nucleotide short fragment of cgcgcgcggac at 106282 and 106290 sites of the wild-type JAK1 gene is deleted, in reference to the sequence of the wild-type JAK1 gene with NCBI accession number NG _ 023402.2. The sequence of 50bp (containing 100 bp) before and after the mutation position of the JAK1 mutant gene, namely the sequence of 106232-106331 site of the JAK1 mutant gene is shown as SEQ ID NO: 1, and the rest sequences are detailed in a reference sequence of a wild-type JAK1 gene with NCBI accession number NG _023402.2, and specifically: the sequence of the 106281 site of the JAK1 mutant gene is the same as the sequence of the same site of a wild type JAK1 gene (NCBI accession number is NG _ 023402.2); the sequence of the JAK1 mutant gene is the same as that of 106290+ N site of the wild JAK1 gene from 106281+ N site (N is an integer between 1 and 135234 in the range of 1-135234). The liver metastasis risk of the male colorectal cancer patient with the JAK1 gene c. [106282 and 106290delcgccgcgac ] mutation is high, so that the mutant gene can be used as a molecular marker of liver metastasis of the male colorectal cancer patient and is applied to construction of a detection kit and a detection method for liver metastasis analysis of the male colorectal cancer patient.
The invention aims to provide a human JAK1 mutant gene, wherein the JAK1 mutant gene is formed by c. [ 238094-. The sequence of 50bp before and after the mutation position of the JAK1 mutant gene, namely the sequence of the 238045-238148 locus (containing 104bp of mutation) of the JAK1 mutant gene is shown as SEQ ID NO: 2, the remaining sequences are detailed in the reference sequence of the wild-type JAK1 gene with NCBI accession number NG _023402.2, specifically: the sequence of the 238094 site of the JAK1 mutant gene is the same as the sequence of the same site of a wild type JAK1 gene (NCBI accession number is NG _ 023402.2), and the sequence of the 238095-238098 site of the JAK1 mutant gene is an insertion mutant sequence TTAT; the sequence of the JAK1 mutant gene is identical to that of 238094+ N site of a wild-type JAK1 gene from 238098+ N site (N is an integer between 1 and 3430). The liver transfer risk of the male colorectal cancer patient with the JAK1 gene having c. [ 238094-.
The object (3) of the present invention is to provide a method for detecting and analyzing a specific JAK1 mutant gene in a male colorectal cancer patient, which comprises the following main steps:
(1) extraction of DNA from male colorectal cancer patient samples
Obtaining a cancer focus tissue or a blood sample of a male 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 JAK1 gene is located
And (2) taking the DNA extracted in the step (1) as a template, and adopting the nucleotide sequence shown as 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 52 ℃ for 20s, extension at 72 ℃ for 30s, and circulation for 30 times; final extension at 72 deg.C for 5 min;
for the c. [238094 and 238095insTTAT ] mutant fragment of JAK1 gene, the sequence 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 54 ℃ for 20s, and extension at 72 ℃ for 40s, and circulating for 30 times; final extension at 72 deg.C for 5 min;
(3) sequencing analysis of JAK1 mutant gene amplification fragment
The JAK1 gene mutation fragment amplified in the step (2) is sent to a gene company for Sanger sequencing, and specific mutation information of a JAK1 gene in a sample is analyzed by referring to a reference sequence of a wild type JAK1 gene with the NCBI accession number of NG _023402.2, so that the JAK1 gene mutation fragment is applied to analysis of liver metastasis of a male colorectal cancer patient. If a c. [106282 and 106290 delcgccgccgac ] mutation of JAK1 gene and/or a c. [238094 and 238095instTAT ] mutation of JAK1 gene are detected in a sample of a male colorectal cancer patient, the result indicates that the patient is liver metastasis or high risk of liver metastasis.
The human JAK1 mutant gene provided by the invention can be used as an effective and reliable molecular marker for liver metastasis of male colorectal cancer, and can also provide clues for mechanism analysis of liver metastasis of colorectal cancer; the JAK1 mutant gene and the detection method thereof can be simply, conveniently and effectively applied to the analysis and prediction of liver metastasis of male colorectal cancer patients, and are beneficial to the early discovery and early intervention of the liver metastasis of the male colorectal cancer patients.
Drawings
FIG. 1 is a partial map of Sanger sequencing before and after the c. [106282-106290 delcgccgccgac ] mutation for the wild-type gene (A) and the mutant gene (B) of JAK 1. 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. [ 238094-. In the figure, the arrow indicates the position where the insertional mutation occurs.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1 the presence of a mutation in the JAK1 specific gene in the vast majority of male colorectal cancer liver metastases
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 patient specific information of 12 families containing at least 1 male patient 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 12 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 200 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 JAK1 gene for 12 families of colorectal cancer is shown in table 1. The results showed that of a total of 24 colorectal cancer patients in 12 families: (1) 13 (92.9%) of 14 male colorectal cancer liver metastasis patients all contained specific mutations of JAK1 gene and the content ratio of the mutations (the percentage of the number of reads of the JAK1 gene specific mutations contained in sequencing effective mutant reads) thereof was more than 3% (3.5% -23%), wherein 7 male colorectal cancer liver metastasis patients, namely, patients 1 and 2 of family #1, patients 1 and 2 of family #2, patients 2 of family #6, patients 1 of family #7 and patients 2 of family #11, all contained c. [ 106282-. (2) No JAK1 gene mutation was detected in any of 4 female colorectal cancer patients. (3) No JAK1 gene mutation was detected in any of 7 patients with colorectal cancer, but not liver metastases. Therefore, JAK1 specific gene mutation, namely c. [106282 and 106290 delcgccgccgac ] or c. [238094 and 238095 instat ] was detected only in the liver metastasis samples of male colorectal cancer, and was found in the vast majority (13 cases/14 cases = 92.9%) of the liver metastasis samples of male colorectal cancer, and further needs to be verified in a large population of patients with colorectal cancer.
Table 112 family information of colorectal cancer and JAK1 gene mutation conditions
Family number | Patient 1 | Mutations and their ratios | Patient 2 | Mutations and their ratios |
#1 | For male, age 56, liver metastasis | c.[106282-106290delcgccgcgac],5.6% | Age 58 in males, liver metastasis | c.[106282-106290delcgccgcgac],15.1% |
#2 | For male, 43 years old, liver metastasis | c.[106282-106290delcgccgcgac],17.6% | For male, 43 years old, liver metastasis | c.[106282-106290delcgccgcgac],18.1% |
#3 | Liver metastasis in 67 years old male | c.[238094-238095insTTAT],23.0% | For male, age 68, liver metastasis | c.[238094-238095insTTAT],3.6% |
#4 | For male, age 65, liver metastasis | c.[238094-238095insTTAT],7.0% | Female, age 66, liver metastasis | - |
#5 | Female, age 58, liver metastasis | - | For male, 59 years old, liver metastasis | c.[238094-238095insTTAT],9.4% |
#6 | Female, age 63, with lung metastasis | - | For male, age 64, liver metastasis | c.[106282-106290delcgccgcgac],6.9% |
#7 | A male, age 60, liver metastasis | c.[106282-106290delcgccgcgac],10.3% | For male, age 65, lung metastasis | - |
#8 | For male, age 46, lung metastasis | - | For male, age 48, liver metastasis | c.[238094-238095insTTAT],4.8% |
#9 | Age 58 in males, liver metastasis | c.[238094-238095insTTAT],3.5% | Age 62 in men with bone metastases | - |
#10 | Male, age 51, lung metastasis | - | For male, age 54, lung metastasis | - |
#11 | A male, age 60, liver metastasis | - | For male, age 62, lung metastasis | c.[106282-106290delcgccgcgac],8.0% |
#12 | For male, age 57, liver metastasis | - | Female, age 58, liver metastasis | - |
Note: "-" indicates no detection.
Example 2 exploration of JAK1 specific Gene mutations in a colorectal cancer patient population
In order to further explore the distribution rule of JAK1 specific gene mutations, namely c. [106282 and 106290delcgccgcgac ] and c. [238094 and 238095instTAT ] in a colorectal cancer patient population, PCR primers for the two specific mutations are designed and 127 postoperative tissue samples of colorectal cancer patients diagnosed with clinical pathology in Fujian province are collected, a sample genome DNA is extracted according to the method in example 1, the extracted genome DNA is used as a template, fragments of the mutations are amplified according to a conventional PCR system and the conditions in Table 2 (each sample is simultaneously and independently amplified by using a primer pair of Seq ID NO: 3 and Seq ID NO: 4 and a primer pair of Seq ID NO: 5 and Seq ID NO: 6), and PCR products are sent to a marine engineering bioengineering company Limited for Sanger sequencing after electrophoresis detection. The results of Sanger sequencing of JAK1 specific gene mutation in 127 colorectal cancer patients are shown in Table 3, and the results show that up to 45 (95.7%) of 47 male colorectal cancer patients with liver metastasis detect specific gene mutation containing JAK1, namely c. [106282 and 106290 delcgcgcgcgcggac ] (Sanger sequencing map at the site of mutation is shown in FIG. 1) or c. [238094 and 238095instTAT ] (Sanger sequencing map at the site of mutation is shown in FIG. 2), while specific mutation of JAK1 gene was not detected in both non-male and non-liver metastatic colorectal cancer patients.
TABLE 2 mutant fragment amplification systems and conditions
Type of mutation | Amplification primers | Amplification conditions |
c.[106282-106290delcgccgcgac] | 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.[238094-238095insTTAT] | 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 JAK1 specific genes in colorectal cancer patient populations
Colorectal cancer patient type | Total number of | c.[106282-106290delcgccgcgac] | c.[238094-238095insTTAT] |
Liver metastasis in men | 47 | 24 | 20 |
Non-hepatic metastasis in males | 20 | 0 | 0 |
Liver metastasis in women | 38 | 0 | 0 |
Non-hepatic metastasis in females | 22 | 0 | 0 |
Summary statistics | 127 | 24 | 20 |
Further, 5 cases of male liver metastasis patients, male non-liver metastasis patients, female liver metastasis patients and female non-liver metastasis patients are selected from 127 cases of colorectal cancer patients, and a primer set designed by a gene delivery company is sent to perform JAK1 whole gene PCR capture sequencing, so that the result shows that the female patients and the non-liver metastasis patients do not have two JAK1 specific gene mutations, and also do not have other mutations of JAK1 genes at other sites and types; the JAK1 gene mutation contained in the male liver transfer patient is mainly two types, namely, c [ 106282-. Therefore, the male colorectal liver transfer patient has one of two specific mutations of JAK1 gene, namely c. [106282 and 106290 delcgcgcgcgcgcgcgcggac ] or c. [238094 and 238095insTTAT ], which are more than 90 percent in total. Therefore, the two specific mutations of the JAK1 gene can be used as molecular markers of liver metastasis of male colorectal cancer patients, and whether the JAK1 gene has the capability of predicting liver metastasis of male colorectal cancer patients needs to be further researched.
Example 3 mutation of JAK1 specific genes can predict liver metastasis in male colorectal cancer patients
30 clinically and pathologically diagnosed male colorectal cancer patients (both feasible resection and impossible resection) in Fujian province are collected, the requirement of first diagnosis of colorectal cancer is met, no metastasis is found in initial diagnosis, other tumors are not combined, 2ml of peripheral venous blood is collected before hospitalization, and 2ml of peripheral venous blood is additionally extracted every 3 months when blood sampling is needed in examination and treatment until clinical images and pathologically diagnosed metastasis of the patients appear. The final effective patients were 25, and another 5 had deaths or missed visits during the study. The 25 patients were treated with an extracellular free nucleic Acid extraction Kit (Qiagen) to extract free nucleic Acid from all blood samples. Two specific mutant fragments of JAK1 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 15 of the metastatic foci were liver, 6 were non-liver (including lung, bone and brain), and 4 were not metastatic in 25 male colorectal cancer patients, and that two specific mutations of JAK1 gene were not detected in 6 non-liver metastatic patients and 4 non-metastatic patients. Without exception, 15 patients with liver metastases contained one of two specific mutations in JAK1 gene, 9 of them contained the c. [106282 and 106290 delcgcgcgcggac ] mutation in JAK1 gene, and 6 contained the c. [238094 and 238095insTTAT ] mutation in JAK1 gene. In 15 cases of male colorectal cancer patients, the specific mutation of JAK1 gene was detected by the molecule earlier than the clinical imaging and/or pathological finding and diagnosis of liver metastasis. Compared with the liver metastasis discovery and diagnosis results of clinical imaging and/or pathology, the detection result of the molecular mutation is 5.2 months earlier (within the range of 3 months to 9 months) on average for the liver metastasis patients of the male colorectal cancer containing the c. [106282 and 106290 delcgcgcgcgcgcgac ] mutation of the JAK1 gene, and is 4.1 months earlier (within the range of 3 months to 6 months) on average for the liver metastasis patients of the c. [238094 and 238095insTTAT ] mutation of the JAK1 gene. It was further shown that the c. [106282 and 106290 delcgccgccgac ] mutation and c. [238094 and 238095 instat ] mutation of the JAK1 gene can be used as molecular markers for liver metastasis in male colorectal cancer patients who had high risk of liver metastasis (9/9 =100% and 6/6 = 100%) in male colorectal cancer patients with two specific gene mutations of the JAK1 gene in blood. 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 425 cases of metastasis in male colorectal cancer patients and their JAK1 specific gene mutations
Transfer type | Number of examples | Contains c. [106282-]Mutation example Number/example | Average advance of molecular detection Time/month | Contains c. [238094-]Mutations Example number/example | Molecular detection mean advance time- Moon cake |
Liver metastasis | 15 | 9 | 5.2 | 6 | 4.1 |
Non-hepatic metastasis | 6 | 0 | - | 0 | - |
Is not transferred | 4 | 0 | - | 0 | - |
Note: "-" indicates no detection.
Sequence listing
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Claims (3)
1. A JAK1 mutant gene, wherein: with reference to the sequence of the wild-type JAK1 gene with NCBI accession number NG _023402.2, the JAK1 mutant gene was formed as a result of a c. [106282 and 106290 delcgcgcgcgcgagc ] deletion mutation in the wild-type JAK1 gene; the sequence of the 106281 site of the JAK1 mutant gene is the same as the sequence of the same site of the wild JAK1 gene; the sequence of the JAK1 mutant gene is the same as the sequence of 106290+ N site of a wild type JAK1 gene from 106281+ N site, and N is an integer between 1 and 135234 within the range of 1-135234; the sequence of 106232-106331 site of JAK1 mutant gene, namely the sequence of 50bp before and after the mutation, is shown as SEQ ID NO: 1 is shown in the specification; the risk of liver metastasis is high in male colorectal cancer patients with JAK1 gene c [106282 and 106290 delcgccgccgac ] mutation.
2. The use of the JAK1 mutant gene of claim 1 in constructing a detection kit and a detection method for liver metastasis analysis of a male colorectal cancer patient.
3. The method for the detection and analysis of JAK1 mutant gene according to claim 1, for the detection of JAK1 mutant gene in male colorectal cancer patients, wherein: the detection method of the JAK1 mutant gene comprises the following main steps:
(1) extraction of DNA from male colorectal cancer patient samples
Obtaining a cancer focus tissue or a blood sample of a male 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 JAK1 gene is located
Taking 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 52 ℃ for 20s, extension at 72 ℃ for 30s, and circulation for 30 times; final extension at 72 deg.C for 5 min;
(3) sequencing analysis of JAK1 mutant gene amplification fragment
The JAK1 gene mutation fragment amplified in the step (2) is sent to a gene company for Sanger sequencing, and specific mutation information of a JAK1 gene in a sample is analyzed by referring to a reference sequence of a wild type JAK1 gene with the NCBI accession number of NG _023402.2, so that the method is applied to analysis of liver metastasis of a male colorectal cancer patient: if a c. [106282-106290 delcgccgccgac ] mutation containing JAK1 gene is detected in a male colorectal cancer patient sample, it indicates that it is liver metastasis or a high risk of liver metastasis.
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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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