CN113621700B - Method for screening red transcription factor EKLF gene mutation and application thereof - Google Patents
Method for screening red transcription factor EKLF gene mutation and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a method for screening red transcription factor EKLF gene mutation and application thereof. The method is characterized in that an amplification primer is designed according to an EKLF gene sequence, and an optimized high-resolution melting curve reaction system is utilized to screen unknown mutation of the EKLF gene in a sample. The detection method provided by the invention has the advantages of simple operation, high speed, high flux, no need of post-treatment of PCR products, capability of detecting the difference of single base, and really realizing closed-tube operation, and wide clinical application prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a method for screening red transcription factor EKLF gene mutation and application thereof.
Background
Erythroid transcription factor EKLF/KLF1 was first discovered by the american medical institute of cinnesa, bieker, which initiates transcription of downstream genes by specific recognition binding to conserved DNA sequences, thereby regulating a series of erythroid growth and development processes. The EKLF gene (nc_ 000019.10) is located at 19p13.2, full length 2781bp, contains three exons, two introns, and the protein consists of 2 functional domains: one at the N-terminus, a transcriptional activation domain enriched by proline; the other is located at the C-terminus and is the zinc finger domain that binds to a conserved DNA sequence. Studies have shown that dominant or double mutations in the EKLF gene can result in a range of blood disease phenotypes, while single allele mutations in the EKLF gene can result in a range of benign phenotypes, such as elevated fetal hemoglobin HbF and adult hemoglobin A2 (HbA 2), and the like, which can further produce phenotypic modifications to the clinical phenotype of b-thalassemia.
Methods for screening unknown mutations of genes mainly include Denaturing High Performance Liquid Chromatography (DHPLC), single Strand Conformation Polymorphism (SSCP), sanger sequencing (gold standard), next generation sequencing, and the like. However, the detection methods have the defects of more or less complicated operation steps, long detection period, higher detection cost and the like. The high resolution melting curve (High resolution meltinganalysis, HRM) technique is a genetic analysis method that can achieve high throughput and reliable results for mutation scanning and genotyping. The main principle of the HRM mutation screening is that the differences exist according to the fragment lengths, GC contents and base complementary pairing of different nucleic acid molecules, so that when a PCR product is denatured by heating, even if the single base changes sufficiently to influence the fragments to cause the difference of melting temperatures (Tm values), double-stranded DNA molecules form unique shapes and positions of melting curves simultaneously, samples are distinguished according to the differences of the melting curves, and finally Sanger sequencing confirmation is carried out on the screened positive samples of the mutation.
Therefore, the application of HRM to screen the EKLF gene mutation has important application value, the current screening method is the Sanger sequencing method most commonly, the experiment is carried out step by step, the required preparation reagent is complex, the time and the labor are consumed, the economic cost is high, the pollution is easy, the method and the detection kit are not suitable for large-scale crowd screening, and therefore, the method and the detection kit for screening the EKLF gene mutation by the large-scale crowd are explored to become hot problems of clinical experimental research.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a detection kit for detecting an unknown mutation of an EKLF gene by using an HRM technology, so as to solve the problems of complex experimental operation steps, long time consumption, easy pollution, high detection cost and the like of the reagent raw materials for detection at present, which are proposed in the background technology, which are prepared in multiple ways.
In order to achieve the above purpose, the present invention provides the following technical solutions.
The invention provides a PCR primer group for detecting EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising 9 pairs of primers in detail as follows:
(1) The upstream and downstream primers of the EKLF gene promoter region are respectively as follows: SEQ ID NO. 1 and SEQ ID NO. 2;
(2) The primers upstream and downstream of the exon 1 region of the EKLF gene are respectively as follows: SEQ ID NO. 3 and SEQ ID NO. 4;
(3) The primers upstream and downstream of the 2-1 region of the exon of the EKLF gene are respectively as follows: SEQ ID NO. 5 and SEQ ID NO. 6;
(4) The primers upstream and downstream of the exon 2-2 region of the EKLF gene are respectively as follows: SEQ ID NO. 7 and SEQ ID NO. 8;
(5) The primers upstream and downstream of the region 2-3 of the exon of the EKLF gene are respectively as follows: SEQ ID NO. 9 and SEQ ID NO. 10;
(6) The primers upstream and downstream of the region 2-4 of the exon of the EKLF gene are respectively as follows: 11 and 12;
(7) The primers upstream and downstream of the region of exon 3 of the EKLF gene are respectively as follows: SEQ ID NO. 13 and SEQ ID NO. 14;
(8) The primers upstream and downstream of the 3' UTR-1 region of the EKLF gene are respectively: 15 and 16;
(9) The primers upstream and downstream of the 3' UTR-2 region of the EKLF gene are respectively: SEQ ID NO. 17 and SEQ ID NO. 18.
The invention also provides a detection reaction system for detecting the EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising one or more PCR primer groups according to claim 1.
Further, the reaction system also comprises negative quality control substances, PCR buffer solution, dNTPs, DNA polymerase, LC Green, betain, deionized water and DMSO; the dNTPs mixture includes dATP, dTTP, dGTP and dCTP.
Further, the DNA polymerase is Taq DNA polymerase of the Guangzhou Ding country organism.
Further, the Betain is 5M.
The invention also provides a detection kit for detecting EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising one or more PCR primer groups according to claim 1.
Further, the kit further comprises the detection reaction system of claim 3.
The invention also provides a detection method for detecting the EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising the following steps:
(1) Preparing DNA of a sample to be tested: collecting a peripheral blood specimen of a detected person, and extracting sample DNA by a column method;
(2) High resolution melting curve reaction system:
the 10 mu LPCR reaction system comprises 10 xTaq buffer (containing 20mM Mg2+) 1.0 mu L,2.5nM dNTPs 0.4 mu L, 5U/. Mu.l Taq DNA polymerase 0.2 mu L,10 mu M upstream and downstream primer 0.6 mu L,1 xLC Green 1 mu L, template DNA0.8 mu L,5MBetain 2.0 mu L, DMSO 1.0 mu L, deionized water 3.0 mu L;
(3) PCR reaction procedure: performing on a PCR instrument according to specific circulation conditions, and pre-denaturing for 5 minutes at 95 ℃;95 ℃ for 30 seconds, 63-68 ℃ for 30 seconds, 72 ℃ for 30 seconds, 45 cycles in total; preserving heat at 95 ℃ for 1 minute and 4 ℃;
(4) Fluorescence was detected on a high resolution melting curve analyzer (LightScanner HR i 96) after PCR amplification was completed, with an initial temperature of 55 ℃, a termination temperature of 98 ℃ and a standby temperature of 42 ℃; the abscissa is temperature and the ordinate is relative fluorescence intensity.
The invention also provides a detection kit for detecting the EKLF gene mutation based on the high-resolution melting curve technology, which is characterized by being applied to detection of the EKLF gene mutation in a sample.
Compared with the prior art, the invention has the beneficial effects.
1) The reaction system of the invention screens unknown mutation of the EKLF gene.
2) The primer designed by the invention has high sensitivity and strong specificity.
3) The method is simple to operate and high in speed, and can screen mutated positive samples in the same day.
4) The PCR product does not need post-treatment, can detect the difference of single base and the like, realizes real tube closing operation, reduces pollution risk and is very suitable for analysis of a large number of samples.
5) The detection system has the advantages of low cost, short period, simple operation, wide popularization in the market and high commercial return.
Drawings
FIG. 1 is a schematic representation of 9 primer pairs covering the EKLF gene region; f represents the forward primer and R represents the reverse primer.
FIG. 2 is a schematic representation of the results of a 9 PCR amplicon HRM screening for partial mutations in the EKLF gene. Different curve shapes of the protrusions represent different variant positive samples.
FIG. 3 is a sequencing diagram of a portion of the HRM screened mutant positive samples for Sanger sequencing validation.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1.
According to the EKLF gene sequence in GenBank, 9 pairs of specific primers are designed by applying Primer express3.0 software gene mutation regions to cover non-coding regions (promoter region, cutting site, 5'UTR and 3' UTR) and all coding regions (exon 1, exon 2 and exon 3) of the EKLF gene, the shortest amplicon fragment length is 179bp, the longest is 348bp, and the specificity of the EKLF gene is primarily identified through BLAST function of NCBI. The positions of the primers are mutually covered, so that the screening dead angle is avoided.
The 9 pairs of upstream and downstream primers are respectively as follows:
1) The upstream and downstream primers of the EKLF gene promoter region are respectively as follows: 5'-TACCCAGCACCTGGACCCTC-3',5'-GAGGCTGTGATAGCCCCTTCG-3';
2) The primers upstream and downstream of the exon 1 region of the EKLF gene are respectively as follows: 5'-CTAAGGACAGAGAGGAGCCC-3', 5'-CAGCCAGCCCACCTAGAC-3';
3) The primers upstream and downstream of the 2-1 region of the exon of the EKLF gene are respectively as follows: 5'-CCAGTGTCCACCGAACCTC-3', 5'-ATCCTCCGAACCCAAAAGCC-3';
4) The primers upstream and downstream of the exon 2-2 region of the EKLF gene are respectively as follows: 5'-CGAGACTCTGGGCGCATA-3', 5'-GGAAGTGCCCTTGGTACTGA-3';
5) The primers upstream and downstream of the region 2-3 of the exon of the EKLF gene are respectively as follows: 5'-GTACCCCGCGATGTACCC-3', 5'-CGGTCTCGGCTATCACACC-3';
6) The primers upstream and downstream of the region 2-4 of the exon of the EKLF gene are respectively as follows: 5'-GGGGACTGCAGAGGATCCA-3', 5'-GCGCCCTTTCTCATGTCC-3';
7) The primers upstream and downstream of the region of exon 3 of the EKLF gene are respectively as follows: 5'-CAGACAGTGGCGCTTATGG-3', 5'-CCCCAGTCACTAGGAGAGTCC-3';
8) The primers upstream and downstream of the 3' UTR-1 region of the EKLF gene are respectively: 5'-CATGAAGCGCCACCTTTGAGC-3',5'-TCTCACTGGGTTTGCACGACA-3';
9) The primers upstream and downstream of the 3' UTR-2 region of the EKLF gene are respectively: 5'-GAGCCACACAGAGATGTCCAAAC-3',5'-TTACAGCCTCCTGCCATCTTCC-3'.
Example 2.
The invention discloses a reaction system containing the PCR primer group and PCR reaction conditions, wherein the reaction system mainly comprises the following components: 9 sets of primer pair mixtures (10. Mu.M), negative quality control, 10 XTaq buffer (containing 20mM Mg2+), 2.5nM dNTPs, 5U/. Mu.l Taq DNA polymerase, 1 XLC Green, template DNA,5M Betain,DMSO and deionized water as described in example 1.
Wherein the reaction conditions are as follows:
1) PCR amplification conditions: pre-denaturation at 95 ℃ for 5 min; 45 cycles (melting: 95 ℃ for 30 seconds, annealing: 63-68 ℃ for 30 seconds, extension: 72 ℃ for 30 seconds); denaturation: the temperature was kept at 95℃for 1 minute and 4 ℃.
2) Reaction conditions for HRM analysis by a resolution melting curve analyzer (LightScanner HR i 96): the Start temperature is 55 ℃, the End temperature is 98 ℃, the standby temperature is 42 ℃, the DNA double strand is gradually denatured and untied in the process that the temperature is gradually increased at 0.1 ℃/s, the LC Green dye falls off from the DNA molecule double strand, the fluorescence value is gradually reduced, the instrument detects the fluorescence value in real time, a melting curve obtained by reducing the fluorescence value along with the temperature increase is established, and the existence of mutation of an unknown sample is judged by comparing the difference of the melting curve between the unknown sequence and a known wild type sequence (negative quality control).
Example 3 HRM detection of EKLF gene mutations was performed by taking EKLF gene promoter region mutation screening as an example.
1) The method specifically comprises the following steps:
step 1, preparing DNA of a sample to be detected: collecting a peripheral blood specimen of a detected person, and extracting sample DNA by a column method;
step 2, split charging reaction liquid: the EKLF gene promoter region upstream and downstream primers and PCR reaction solution were mixed and split into 96-well PCR plates of Bio-Rad, each well comprising 10×Taq buffer (containing 20mM Mg2+) 1.0. Mu.L, 2.5nM dNTPs 0.4. Mu.L, 5U/. Mu.l Taq DNA polymerase 0.2. Mu.L, 10. Mu.M upstream and downstream primers 0.6. Mu.L, 1×LC Green 1. Mu.L, template DNA 0.8. Mu.L, 5M Betain 2.0. Mu.L, DMSO 1.0. Mu.L, deionized water 3.0. Mu.L;
step 3, adding DNA of a sample to be detected: adding 1 mu L of negative quality control material into the first three holes of a 96-hole PCR plate, adding 1 mu L of different sample DNA to be detected into the other 96 holes, performing instantaneous centrifugation on the 96-hole plate to ensure that the DNA is mixed into a reaction solution, performing liquid sealing by liquid paraffin, and finally covering the upper part of the 96-hole PCR plate by using tin foil;
step 4, PCR reaction: performing pre-denaturation at-95 ℃ for 5 minutes on a PCR instrument according to specific circulation conditions; 45 cycles (melting: 95 ℃ for 30 seconds, annealing: 63-68 ℃ for 30 seconds, extension: 72 ℃ for 30 seconds); denaturation: preserving heat at 95 ℃ for 1 minute and 4 ℃;
step 5, hrm analysis: after the PCR amplification was completed, fluorescence was detected on a high resolution melting curve analyzer (LightScanner HR I96), with an initial temperature "Start Temp" of 55 ℃, an End temperature "End Temp" of 98℃and a standby temperature "Hold Temp" of 42 ℃;
step 6, result interpretation: and selecting positive samples according to a melting curve scanned by the HRM instrument.
2) Results: the HRM analysis results are shown in fig. 3. DNA sequencing is carried out on the detected positive sample, NCBI Blast is used for comparison, and the comparison result is consistent with the result of analyzing a high-resolution melting curve by using a high-resolution melting curve analyzer (LightScanner HR I96), so that the detection method provided by the invention can effectively detect the EKLF mutant genes.
3) Conclusion: the invention detects the EKLF gene mutation by using a high-resolution melting curve for the first time, and expands the application of EKLF gene mutation screening. The kit is simple to operate, high in speed, and capable of detecting single base difference without post-treatment of PCR products, truly realizing tube closing operation and detecting EKLF gene mutation.
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cggtctcggc tatcacacc 19
<210> 11
<211> 19
<212> DNA
<213> artificial sequence
<400> 11
ggggactgca gaggatcca 19
<210> 12
<211> 18
<212> DNA
<213> artificial sequence
<400> 12
gcgccctttc tcatgtcc 18
<210> 13
<211> 19
<212> DNA
<213> artificial sequence
<400> 13
cagacagtgg cgcttatgg 19
<210> 14
<211> 21
<212> DNA
<213> artificial sequence
<400> 14
ccccagtcac taggagagtc c 21
<210> 15
<211> 21
<212> DNA
<213> artificial sequence
<400> 15
catgaagcgc cacctttgag c 21
<210> 16
<211> 21
<212> DNA
<213> artificial sequence
<400> 16
tctcactggg tttgcacgac a 21
<210> 17
<211> 23
<212> DNA
<213> artificial sequence
<400> 17
gagccacaca gagatgtcca aac 23
<210> 18
<211> 22
<212> DNA
<213> artificial sequence
<400> 18
ttacagcctc ctgccatctt cc 22
Claims (9)
1. A PCR primer set for detecting EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising 9 pairs of primers, and is characterized in that the following details are:
(1) The upstream and downstream primers of the EKLF gene promoter region are respectively as follows: SEQ ID NO. 1 and SEQ ID NO. 2;
(2) The primers upstream and downstream of the exon 1 region of the EKLF gene are respectively as follows: SEQ ID NO. 3 and SEQ ID NO. 4;
(3) The primers upstream and downstream of the 2-1 region of the exon of the EKLF gene are respectively as follows: SEQ ID NO. 5 and SEQ ID NO. 6;
(4) The primers upstream and downstream of the exon 2-2 region of the EKLF gene are respectively as follows: SEQ ID NO. 7 and SEQ ID NO. 8;
(5) The primers upstream and downstream of the region 2-3 of the exon of the EKLF gene are respectively as follows: SEQ ID NO. 9 and SEQ ID NO. 10;
(6) The primers upstream and downstream of the region 2-4 of the exon of the EKLF gene are respectively as follows: 11 and 12;
(7) The primers upstream and downstream of the region of exon 3 of the EKLF gene are respectively as follows: SEQ ID NO. 13 and SEQ ID NO. 14;
(8) The primers upstream and downstream of the 3' UTR-1 region of the EKLF gene are respectively: 15 and 16;
(9) The primers upstream and downstream of the 3' UTR-2 region of the EKLF gene are respectively: SEQ ID NO. 17 and SEQ ID NO. 18.
2. A detection reaction system for detecting EKLF gene mutation based on a high-resolution melting curve technology, which is characterized by comprising the PCR primer group of 9 groups according to claim 1.
3. The detection reaction system of claim 2, wherein the reaction system further comprises a negative quality control, PCR buffer, dNTPs, DNA polymerase, LC Green, betain, deionized water, and DMSO; the dNTPs mixture includes dATP, dTTP, dGTP and dCTP.
4. The detection reaction system according to claim 3, wherein the DNA polymerase is Taq DNA polymerase of the tripod China organism.
5. The detection reaction system according to claim 3, wherein the Betain concentration is 5M.
6. A detection kit for detecting EKLF gene mutation based on high resolution melting curve technology, characterized in that the kit contains the PCR primer set of 9 sets according to claim 1.
7. The test kit according to claim 6, further comprising the test reaction system according to claim 3.
8. A method for detecting EKLF gene mutations based on high resolution melting curve technology for non-diagnostic and therapeutic purposes, comprising the steps of:
(1) Preparing DNA of a sample to be tested: collecting a peripheral blood specimen of a detected person, and extracting sample DNA by a column method;
(2) High resolution melting curve reaction system:
the 10. Mu.LPCR reaction system comprises 10 XTaq buffer 1.0. Mu.L, 2.5nM dNTPs 0.4. Mu.L, 5U/. Mu.l Taq DNA polymerase 0.2. Mu.L, 10. Mu.M upstream and downstream primer 0.6. Mu.L, 1 XLC Green 1. Mu.L, template DNA 0.8. Mu.L, 5MBetain 2.0. Mu.L, DMSO 1.0. Mu.L, deionized water 3.0. Mu.L; the 10 xTaq buffer is 20mM Mg 2+ ；
(3) PCR reaction procedure:
performing on a PCR instrument according to specific circulation conditions, and pre-denaturing for 5 minutes at 95 ℃;95 ℃ for 30 seconds, 63-68 ℃ for 30 seconds, 72 ℃ for 30 seconds, 45 cycles in total; preserving heat at 95 ℃ for 1 minute and 4 ℃;
(4) Detecting fluorescence on a high-resolution melting curve analyzer with the model of LightScanner HR I96 after the PCR amplification is finished, wherein the initial temperature is 55 ℃, the end temperature is 98 ℃ and the standby temperature is 42 ℃; the abscissa is temperature and the ordinate is relative fluorescence intensity.
9. Use of a kit according to claim 6 or 7 for the detection of EKLF gene mutations in samples for non-diagnostic and therapeutic purposes.
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