CN110982887A - Primer for LKB1 gene mutation detection and application thereof - Google Patents
Primer for LKB1 gene mutation detection and application thereof Download PDFInfo
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Abstract
The invention relates to a primer for LKB1 gene mutation detection and application thereof. The invention designs a wild type primer and a mutant type primer of LKB1 exon 8 (LKB1-E8), respectively amplifies corresponding wild type target fragments and mutant type target fragments, mixes the two fragments in different proportions to ensure that the content of the mutant type target fragments is 1/10, 1/100, 1/1000 and 0, and finally distinguishes by an HRM method; compared with the prior art, the invention has the advantages of high specificity, high sensitivity, convenience and rapidness; and the flux is higher, and the single cost is lower.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a primer for LKB1 gene mutation detection and application thereof.
Background
The human LKB1(Liver Kinase B1) gene or STK11 (spring-Threonine Kinase 11, STK11) is located on human chromosome 19p 13.3. Contains 10 exons, encodes protein LKB1 which consists of 433 amino acids, has a molecular weight of about 50kda, and comprises a kinase region (44-309), an N-terminal regulatory domain and a C-terminal regulatory domain. The N-terminal regulatory domain contains a nuclear localization sequence that allows LKB1 to localize in the nucleus. LKB1 is widely expressed in various tissues of the human body. With the largest presence of immature liver, testis, small intestine and skeletal muscle.
Germline inactivating mutations in the LKB1 gene can lead to the susceptibility of the cancer to the patient to pigerman syndrome (Peutz-Jegherssyndrome, PJ S), who has multiple hamartoma polyps and an increased risk of developing cancer. Somatic mutations in the LKB1 gene are widely present in many types of malignancies, such as lung, colon, and breast cancers, and therefore LKB1 is widely recognized as a cancer suppressor gene. The LKB1 protein, which is the encoded product of the LKB1 gene, is a serine/threonine kinase that regulates a variety of cellular physiopathological processes.
Direct substrates for LKB1 include AMPK (AMP-activated protein kinase) and twelve AMPK kinases LKB1 enhances AMPK phosphorylation by promoting phosphorylation at the Thr172 site on the AMPK α subunit, thereby activating AMPK.
LKB1 can inhibit the activity of the eukaryotic cell growth positive regulator mTORC1(mammaliantarget of rapamycin complex 1) by activating AMPK, whereas mTORC1 can promote cell growth and cell cycle progression. In many tumor cells, the activity of mTORC1 is abnormally activated.
After AMPK is activated by LKB1, AMPK rapidly inactivates acetyl-coa carboxylase (ACC), which is a rate-limiting enzyme for fatty acid and cholesterol synthesis, and HMG-coa reductase (HMGCR), thereby inhibiting lipid synthesis and reducing energy consumption. Meanwhile, the activity of glycolytic key enzyme 6-phosphofructokinase is rapidly regulated to promote glycolysis to generate energy. Enzymes such as ACC are essential for the survival of some tumor cells, and chemical inhibition of their activity inhibits the growth of cancer and prostate cancer transplants.
Mutations in the LKB1 gene site are associated with the development of many tumors, including lung, colon, and breast cancers, and are highly valued as being predictive of malignant transformation of cells and possible tumor development. Therefore, the detection of the LKB1 gene site mutation plays a very important role.
As is well known, the high-resolution melt curve (HRM) analysis technique is a recent genetic analysis method for mutation scanning and genotyping that has emerged abroad in recent years. It is a high-efficiency and steady PCR technology, is not limited by mutation base site and type, does not need sequence specific probe, and is directly used after PCR (polymerase chain reaction), namely polymerase chain reactionAnd (3) running high-resolution melting to complete analysis of sample mutation, single nucleotide polymorphism-SNP, methylation, match and the like. Because the operation is simple, convenient and quick, the use cost is low, the result is accurate, the real pipe closing operation is realized, and the HRM technology is generally concerned. High resolution melting curves (HRMs) are, however, extensions of conventional melting curve analysis: it requires special fluorescent dye, high-performance fluorescent quantitative PCR and special analysis algorithm; so that the PCR amplification products can be directly screened for the existence of genetic variations (SNPs) without sequencing. SYBRTMGreen I is toxic to PCR amplification, so it is necessary to use low concentrations of dye, and because it binds in a non-saturated state, the dye can recombine during melting, making it unsuitable for HRM.
At the same time, roche developed a new dye resplight or LC Green suitable for HRM, which has three advantages: 1. the saturated dye has low toxicity; 2. the concentration can make the DNA reach saturation; 3. the potential for dye site recombination is reduced. The requirement of HRM of high resolution melting curve of PCR (polymerase Chain reaction) product is that the instrument is high resolution and homogeneous instrument, which ensures that the result difference is only influenced by DNA template; the reagent is stable and reliable PCR and HRM dye, and ensures to obtain a high-resolution melting curve; software, high resolution melting curve analysis special software. The LightCycler Nano System of the Roche fluorescence quantitative PCR System has the advantages of good light source, good temperature control, good detection and fast heating, can completely meet the detection requirement, passes the examination and approval of the national drug administration in the near term, and is suitable for clinical diagnosis and detection. The lightcyclenano System is a scaled down version of the LightCycler 480 System,
480 published in the HRM field is the most widely published real-time quantitative PCR system currently: the application fields comprise more than 10 influencing factors of human diseases, plants, microorganisms, viruses, pharmacology, toxicology, physiology, diagnosis and the like, and more than 5 influencing factors comprise more than 10 articles of Nature, Nature Methods, Nature genetics, and the influencing factors of 5-10 and 10 articles of more than 5 articles, wherein the total number of the articles accounts for 18 percent, and the increasing trend is obvious year by year, and the qPCR amplification has become the current qPCR amplificationThe hot spots for the development of applications currently often use sequencing methods to detect the presence of mutations, but to summarize, the existing sequencing methods have three disadvantages:
firstly, the method comprises the following steps: low sensitivity, low proportion of mutations (< 20%) were undetectable;
secondly, the method comprises the following steps: the time consumption is long, and generally 2 to 3 days are needed;
thirdly, the method comprises the following steps: the cost is high.
In view of the above problems, a rapid detection method for LKB1 gene mutation, which has high sensitivity, short time consumption, low cost, and higher operability, and the development of primers for LKB1 gene mutation detection are indispensable.
Disclosure of Invention
The invention aims to provide a primer for LKB1 gene mutation detection and application thereof. Based on the primer for LKB1 gene mutation detection, LKB1 gene mutation can be rapidly detected, so that the defects of the existing sequencing method are overcome: firstly, the method comprises the following steps: low sensitivity, low proportion of mutations (< 20%) were undetectable; secondly, the method comprises the following steps: the time consumption is long, and generally 2 to 3 days are needed; thirdly, the method comprises the following steps: the cost is high.
The purpose of the invention can be realized by the following technical scheme:
the invention firstly provides primers for LKB1 gene mutation detection, which comprise wild type primers and mutant type primers of LKB1 No. 8 exon (LKB 1-E8);
wherein, the forward primer sequence of the wild type primer of the LKB1 No. 8 exon is shown as SEQ ID NO.1,
the reverse primer sequence of the wild type primer of the LKB1 exon 8 is shown in SEQ ID NO.2,
the sequence of the forward primer of the mutant primer of LKB1 exon 8 is shown as SEQ ID NO.3,
the reverse primer sequence of the mutant primer of LKB1 exon 8 is shown in SEQ ID NO. 4.
The invention also provides application of the primer for LKB1 gene mutation detection in preparation of a detection reagent for detecting LKB1 gene mutation or a detection kit for detecting LKB1 gene mutation.
The invention also provides a rapid detection method of LKB1 gene mutation for non-diagnosis purposes by using the primer for LKB1 gene mutation detection, which comprises the following steps:
respectively designing a pair of wild primers aiming at two ends of a LKB1 No. 8 exon mutation site, and designing a pair of mutation primers on the mutation site;
respectively amplifying a wild type template and a mutant fragment, carrying out PCR amplification by using a wild type primer and using the two mutant fragments as templates to obtain a mutant template, and amplifying the corresponding mutant template;
wild-type and mutant templates were mixed at different ratios and distinguished by HRM.
The high resolution melting curves of the PCR products are based on the difference in genotyping-Tm of HRM, and the Tm of the PCR products depends on GC content.
High Tm G: C > a: T > G: G > G: T ═ G: a > T: T ═ a: a > T: C > a: C low Tm.
Melting curves are carried out on the amplification products of the homozygous sub-samples to obtain melting curves with similar peak shapes, and melting curves are carried out on the amplification products of the heterozygous sub-samples to obtain melting curves with different peak shapes;
wherein the homozygote comprises a wild-type homozygote or a mutant homozygote.
The different proportions of the wild-type template and the mutant template refer to that: the mutant templates were made to contain 1/10, 1/100, 1/1000 and 0 and finally distinguished by the HRM method.
Specifically, the primer for LKB1 gene mutation detection is used for a rapid detection method of LKB1 gene mutation for non-diagnostic purposes, and comprises the following steps:
sample treatment: taking 2ml of peripheral blood, placing in an EDTA anticoagulation tube, slightly reversing and uniformly mixing, and storing at 4 ℃;
DNA extraction: extracting DNA with 200 μ L of anticoagulation kit, and detecting DNA purity and concentration by electrophoresis gel imaging;
qPCR-HRM assay comprising: setting up control wells and detecting duplicate wells; 10 mu L of reaction system; adding the reagents into the eight-connected tube in sequence, and uniformly mixing; after all samples are mixed, placing the eight-connected tube in a Roche fluorescence quantitative PCR system instrument for procedural detection;
and (3) analyzing and judging results: in the high-resolution melting curve analysis method, a sample well differing from a base line by more than a positive control well is judged as a mutant type, and a sample well differing from the base line by less than the positive control well is judged as a wild type, with the positive control well as a boundary.
The qPCR-HRM detection procedure comprises: denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10 seconds; annealing is carried out in a bottom detection mode, the bottom detection annealing temperature is set to be 55-65 ℃, the temperature is reduced by 1 ℃ in each cycle, and the time is 10 seconds; extension at 72 ℃ for 30 seconds; repeating the steps 2 to the fourth step for 45 cycles; denaturation at 95 ℃ for 60 seconds; denaturation at 40 ℃ for 60 seconds; the high resolution melting temperature was set from 60 ℃ to 95 ℃ with a ramp rate of 0.05 ℃/s.
Obtaining LKB1 exon 8 mutant templates comprising the steps of:
obtaining two sections of mutant fragments before and after a mutation site by using a mutation primer with the mutation site;
these two mutant fragments were ligated to form a LKB1 exon 8 mutant template.
Obtaining wild-type template of LKB1 No. 8 exon, comprising the steps of:
performing an experiment by using a wild type primer of LKB1 No. 8 exon by using a known unmutated genome as a template to obtain a specific wild type template with a single band; the wild type template is diluted to 1 ten thousand to 10 ten thousand times and is used as a negative control template for detecting the wild type.
Compared with the prior art, the invention has the beneficial effects that:
the method respectively designs a wild type primer and a mutant type primer of an LKB1 No. 8 exon (LKB1-E8), respectively amplifies corresponding wild type target fragments and mutant type target fragments, mixes the two fragments in different proportions, enables the content of the mutant type target fragments to be 1/10, 1/100, 1/1000 and 0, and finally distinguishes by an HRM method, and adopts a high-resolution melting curve method (HRM method). compared with other methods, the HRM method can detect 0.1% of mutation, the detection time only needs 1 hour, and the reagent consumable cost per sample is less than ￥ 7.00.00.
Drawings
FIG. 1 is a schematic diagram of a functional domain of LKB1 protein and a hot-spot mutation site according to the invention;
FIG. 2 is a HRM plot of LKB1 exon 8 (LKB1-E8)1/10, 1/100, 1/1000 mutant and wild-type templates tested using wild-type primers.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Examples
The invention is applied to the fields of biotechnology and medicine, in particular to the technologies of molecular biology, molecular diagnosis and real-time quantitative PCR.
Referring to fig. 1-2, the present invention designs wild-type primers and mutant primers for exon 8 of LKB1 (LKB1-E8), respectively, amplifies corresponding target fragments of wild-type and mutant, respectively, mixes the two fragments in different ratios to make the content of target fragments of mutant 1/10, 1/100, 1/1000 and 0, and finally distinguishes them by HRM method. The following will explain the specific operation by taking LKB1-E8 as an example.
1. Sample treatment: 2ml of peripheral blood is taken out and put into an EDTA anticoagulation tube, and the mixture is gently inverted and mixed evenly and stored at 4 ℃.
2. DNA extraction: 200. mu.L of anticoagulated DNA was extracted with QIAmp DNAblood Mini Kit. The purity and concentration of DNA were checked by electrophoresis gel imaging.
3. qPCR-HRM detection system:
1) control set-up and assay replicate wells
Each sample must be tested simultaneously with control experiments, including a wild-type negative control and an m/w (mutation/wild-1%) positive control. Duplicate wells were used for both control and sample.
2)10 μ L of the reaction system (primer sequences are shown in the attached Table)
Sample addition order reagent volume (μ L)
6 HRM mix 5
2 25mM Mgcl2 1.2
3 primer LKB 1-E8F 0.2
4 primer LKB 1-E8R 0.2
5 template 0.5
1 H2O 3.2
3) Adding the reagents into the eight-connected tube in sequence, mixing uniformly, and covering the eight-connected tube with a cover.
4) After all samples were mixed, place the eight tubes in Roche fluorescence quantitative PCR System-LightCycler Nano instrument, take care that group A and group D in the instrument are balanced, close the instrument lid.
4. Procedure for qPCR-HRM detection:
1) denaturation at 95 ℃ for 10 min;
2) denaturation at 95 ℃ for 10 seconds;
3) annealing is carried out in a bottom detection mode, the bottom detection annealing temperature is set to be 55-65 ℃, the temperature is reduced by 1 ℃ in each cycle, and the time is 10 seconds;
4) extension at 72 ℃ for 30 seconds;
5) repeating the steps 2 to the fourth step for 45 cycles;
6) denaturation at 95 ℃ for 60 seconds;
7) denaturation at 40 ℃ for 60 seconds;
8) the high resolution melting temperature was set from 60 ℃ to 95 ℃ with a ramp rate of 0.05 ℃/s.
5. And (4) analyzing results:
1) the sample name, the detection target and the fluorescent dye used are set.
2) The specificity of PCR bands of each hole can be checked through the melting curve graph of each hole; the wild-type sample wells are used as baselines, and the positive control well and sample well maps can be checked through a high-resolution melting curve.
3) Determination of results
In the high-resolution melting curve analysis method, a sample well differing from the base line by more than the positive control well is judged as a mutant type and a sample well differing by less than the positive control well is judged as a wild type, using the positive control well as a boundary (see fig. 2).
4) And acquiring a result graph.
6. Obtaining a wild type target fragment:
experiments are carried out by taking a known unmutated genome as a template and LKB 1-E8F + LKB 1-E8R as primers, and specific wild type target fragments with single bands are obtained by referring to the reaction systems and the procedures of the steps 3 and 4.
The target fragment is diluted to 1 ten thousand to 10 ten thousand times, and the final concentration is about 10 ng/. mu.L, and the target fragment is used as a negative control template for detecting wild type.
7. Obtaining LKB1-E8 mutant target fragments:
obtaining a mutant target fragment by two steps, wherein in the first step, a mutant primer (LKB1-E8V320L F, LKB1-E8V320L R) with a mutation site is used for obtaining two sections of mutant fragments before and after the mutation site; in the second step, the two mutant fragments are connected to form the objective LKB1-E8 mutant.
1) Mutant first step PCR
Using the diluted target fragment of wild type as a template, and respectively
Two groups of experiments are carried out by taking LKB 1-E8F + LKB1-E8V320L R, LKB1-E8V320L F + LKB 1-E8R as primers, and the experimental system and the program refer to the steps 3 and 4 to obtain a single fragment of two strips.
2) Mutation first step product dilution
The two PCR products obtained in the first step of mutagenesis were diluted 1 to 10 ten thousand times to a final concentration of about 10 ng/. mu.L.
3) Mutant second step PCR
And (3) performing second-step PCR by using the diluted first-step two-section products as templates and LKB 1-E8F + LKB 1-E8R as primers, wherein the experimental system and the program refer to the steps 3 and 4, and obtaining a single-strip fragment.
4) Mutating second step product dilution
The PCR product obtained in the second step of mutation was diluted 1 to 10 ten thousand times to a final concentration of about 10 ng/. mu.L.
8. Acquisition of M/W-1/10, 1/100, 1/1000 positive control template:
mixing 9 μ L of the wild-type template and 1 μ L of the mutant template to prepare 10 μ L of a mixed template of mutant/wild-type (M/W) ═ 1/10 as a 1/10 positive control template for detection; this template was diluted 10-fold with a wild-type template to obtain mixed templates of mutant/wild-type (M/W) 1/100 and 1/1000 as positive control templates for detection 1/100 and 1/1000, respectively.
9. And (4) analyzing results:
the high specificity and high sensitivity of the method were demonstrated by the ability to distinguish between templates containing the 1/1000 mutant by the HRM method, and the total run time required was only 1 hour.
Attached table: LKB1-E8V320L F, LKB1-E8V320L R detection primer sequences
Compared with other methods, the technology of the invention has the advantages of high specificity, high sensitivity, convenience and rapidness, higher flux and lower single cost.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> Shanghai health medical college
<120> primer for LKB1 gene mutation detection and application thereof
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ctgaagcacc attgcccatc ccac 24
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gtgggatggg caatggtgct tcag 24
Claims (10)
1. Primers for LKB1 gene mutation detection, which are characterized by comprising wild type primers and mutant type primers of LKB1 exon 8 (LKB 1-E8);
wherein, the forward primer sequence of the wild type primer of the LKB1 No. 8 exon is shown as SEQ ID NO.1,
the reverse primer sequence of the wild type primer of the LKB1 exon 8 is shown in SEQ ID NO.2,
the sequence of the forward primer of the mutant primer of LKB1 exon 8 is shown as SEQ ID NO.3,
the reverse primer sequence of the mutant primer of LKB1 exon 8 is shown in SEQ ID NO. 4.
2. Use of the primers for LKB1 gene mutation detection of claim 1 in the preparation of a detection reagent for LKB1 gene mutation or a detection kit for LKB1 gene mutation.
3. Method for the rapid detection of mutations in LKB1 gene for non-diagnostic purposes based on the primers for LKB1 gene mutation detection claimed in claim 1, characterized in that it comprises the following steps:
respectively designing a pair of wild primers aiming at two ends of a LKB1 No. 8 exon mutation site, and designing a pair of mutation primers on the mutation site;
respectively amplifying a wild type template and a mutant fragment, carrying out PCR amplification by using a wild type primer and using the two mutant fragments as templates to obtain a mutant template, and amplifying the corresponding mutant template;
wild-type and mutant templates were mixed at different ratios and distinguished by HRM.
4. The method of claim 3, wherein the high resolution melting curve of the PCR product is based on the difference of the genotyping-Tm of the HRM, and the Tm of the PCR product is dependent on the GC content.
5. The method of claim 3, wherein the amplification products of the homozygous samples are subjected to melting curves to obtain melting curves with similar peak shapes, and the amplification products of the heterozygous samples are subjected to melting curves to obtain melting curves with different peak shapes;
wherein the homozygote comprises a wild-type homozygote or a mutant homozygote.
6. The method of claim 3, wherein the different ratios of the wild-type template and the mutant template are: the mutant templates were made to contain 1/10, 1/100, 1/1000 and 0 and finally distinguished by the HRM method.
7. A method according to claim 3, characterized in that it comprises the following steps:
sample treatment: taking 2ml of peripheral blood, placing in an EDTA anticoagulation tube, slightly reversing and uniformly mixing, and storing at 4 ℃;
DNA extraction: extracting DNA with 200 μ L of anticoagulation kit, and detecting DNA purity and concentration by electrophoresis gel imaging;
qPCR-HRM assay comprising: setting up control wells and detecting duplicate wells; 10 mu L of reaction system; adding the reagents into the eight-connected tube in sequence, and uniformly mixing; after all samples are mixed, placing the eight-connected tube in a Roche fluorescence quantitative PCR system instrument for procedural detection;
and (3) analyzing and judging results: in the high-resolution melting curve analysis method, a sample well differing from a base line by more than a positive control well is judged as a mutant type, and a sample well differing from the base line by less than the positive control well is judged as a wild type, with the positive control well as a boundary.
8. The method of claim 7, wherein the qPCR-HRM detection procedure comprises: denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10 seconds; annealing is carried out in a bottom detection mode, the bottom detection annealing temperature is set to be 55-65 ℃, the temperature is reduced by 1 ℃ in each cycle, and the time is 10 seconds; extension at 72 ℃ for 30 seconds; repeating the steps 2 to the fourth step for 45 cycles; denaturation at 95 ℃ for 60 seconds; denaturation at 40 ℃ for 60 seconds; the high resolution melting temperature was set from 60 ℃ to 95 ℃ with a ramp rate of 0.05 ℃/s.
9. The method of claim 7, wherein an exon 8 mutant template for LKB1 is obtained, comprising the steps of:
obtaining two sections of mutant fragments before and after a mutation site by using a mutation primer with the mutation site;
these two mutant fragments were ligated to form a LKB1 exon 8 mutant template.
10. The method of claim 7,
obtaining wild-type template of LKB1 No. 8 exon, comprising the steps of:
performing an experiment by using a wild type primer of LKB1 No. 8 exon by using a known unmutated genome as a template to obtain a specific wild type template with a single band; the wild type template is diluted to 1 ten thousand to 10 ten thousand times and is used as a negative control template for detecting the wild type.
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|---|---|---|---|---|
| CN1982477A (en) * | 2005-12-13 | 2007-06-20 | 居里研究所 | Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids |
| CN103436593A (en) * | 2012-09-11 | 2013-12-11 | 上海赛安生物医药科技有限公司 | Rapid detection method for p53 gene mutation |
| CN103525901A (en) * | 2012-09-11 | 2014-01-22 | 上海赛安生物医药科技有限公司 | Rapid detection method for MEK1 gene mutation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982477A (en) * | 2005-12-13 | 2007-06-20 | 居里研究所 | Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids |
| CN103436593A (en) * | 2012-09-11 | 2013-12-11 | 上海赛安生物医药科技有限公司 | Rapid detection method for p53 gene mutation |
| CN103525901A (en) * | 2012-09-11 | 2014-01-22 | 上海赛安生物医药科技有限公司 | Rapid detection method for MEK1 gene mutation |
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| WENDY LIM ET AL.: "Relative Frequency and Morphology of Cancers in STK11 Mutation Carriers", 《GASTROENTEROLOGY》 * |
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Application publication date: 20200410 |