阅读说明：本技术 用于检测ak4基因多态性的探针、引物、试剂盒 (Probe, primer and kit for detecting AK4 gene polymorphism ) 是由 傅咏南 沈勋章 张奕 毛丹丹 于 2019-10-12 设计创作，主要内容包括：本发明公开了一种用于检测AK4基因多态性的探针、引物、试剂盒,其中探针的序列如SEQ ID NO.:1、SEQ ID NO.:4、或SEQ ID NO.:7所示,或者在SEQ ID NO.:1、SEQ ID NO.:4、SEQ ID NO.:7的序列两端加以基团修饰。本发明的用于检测AK4基因多态性的探针、引物、试剂盒,在检测AK4基因多态性时,具有灵敏度高、特异性好、快速、高通量检测等优点。(The invention discloses a probe, a primer and a kit for detecting AK4 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7, or both ends of the sequence of the SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7 are modified by groups. The probe, the primer and the kit for detecting the AK4 gene polymorphism have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when detecting the AK4 gene polymorphism.)

1. A probe for detecting AK4 gene polymorphism, wherein the sequence of the probe is shown as SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7; or the two ends of the sequences of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 7 are modified by groups.

2. The probe of claim 1, wherein the modifying group of SEQ ID No. 1 is: the modifying group of 5' is a fluorescent group HEX; the modifying group of the 3' is a quenching group BHQ 1; wherein the modifying group of SEQ ID No. 4 is: the modifying group of 5' is a fluorescent group FAM; the modifying group of the 3' is a quenching group BHQ 1; wherein the modifying group of SEQ ID No. 7 is: the modification group of 5' is a fluorescent group CY 5; the modifying group at the 3' is a quenching group BHQ 2.

3. The probe of claim 1, wherein the probe comprises a loop sequence and a stem sequence, wherein the loop sequence of SEQ ID No. 1 is 6-26 bp; wherein the loop sequence of SEQ ID NO. 4 is 6-16 bp; wherein the loop sequence of SEQ ID NO. 7 is 6-18 bp.

4. A primer for detecting AK4 gene polymorphism, which is characterized in that the sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3; 5 and 6 in SEQ ID No.: 5; SEQ ID No. 8 and SEQ ID No. 9.

5. Kit for detecting the polymorphism of AK4 gene, characterized in that it comprises the probe according to any one of claims 1 to 3 and/or the primer according to claim 4.

6. The kit of claim 5, wherein the kit further comprises Taq enzyme, dNTP, and/or Mg2+, and instructions for use.

7. A method for detecting an AK4 gene polymorphism, comprising the steps of:

(1) collecting a sample and extracting DNA;

(2) performing a fluorescent quantitative PCR reaction using the probe of any one of claims 1 to 3 and the primer of claim 4;

(3) and (3) analyzing the result by using a PCR instrument matched software, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks.

Technical Field

The invention relates to the field of molecular biology, in particular to a probe, a primer and a kit for detecting AK4 gene polymorphism.

Background

Adenylate Kinase (AK) family isozymes are widely present in organisms, are the machinery of intracellular nucleotide synthesis, and are involved in intracellular energy and nucleotide metabolism. They reversibly catalyze the transfer of phosphate groups between ATP and AMP and release 2 molecules of ADP. The AK4 gene is localized at 1p31, and the enzyme is mainly expressed in tissues rich in mitochondria such as brain, heart, kidney and liver. AK4 can catalyze phosphorylation of dAMP, CMP and dCMP with ATP or GTP as phosphate donors, and phosphorylate AMP with UTP as phosphate donor. Studies have shown that AK4 can regulate adenosine homeostasis, thereby affecting cell survival and proliferation. The expression of the AK4 gene in a human liver cancer cell line (HepG 2) and a human embryonic kidney cell (HEK 293) which are cultured in a low-oxygen environment is in opposite trends, and the fact that the expression of the AK4 gene has a protective effect on low-oxygen stress cells is suggested.

Although defects in AKs are known to cause some specific diseases, little is known about complex diseases caused by low AK activity, such as degenerative diseases. ATP is the most important molecule carrying high energy, and when cells play different functions, the amount of ATP produced is different, and the whole energy deficiency can cause various organ failures. By researching energy metabolism related enzymes and better understanding of a phosphate group transfer mechanism, a solid theoretical basis can be provided for the future development and treatment of diseases caused by energy metabolism disorder.

The genetic polymorphism forms the genetic basis of different individual susceptibility to diseases and different responses to drugs and environmental factors, and the selection of the appropriate genetic polymorphism is very important for understanding the nature of the phenotype on the genetic level.

The currently widely applied gene polymorphism detection method is a direct DNA sequencing method, and the direct DNA sequence analysis of a PCR product can define mutation sites, but has the defects of time and labor waste, high cost, inapplicability to the detection of a large number of samples and the like. Therefore, it is necessary to develop a method suitable for the detection of large-scale samples.

Disclosure of Invention

The invention aims to provide 3 groups of probes for detecting AK4 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID NO. 1, SEQ ID NO. 4 or SEQ ID NO. 7; or the two ends of the sequences of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 7 are modified by groups;

wherein the modifying group is: the 5' modifying group is a fluorescent group, such as FAM, HEX, CY 5; the modifying group of 3' is a quenching group, such as BHQ1 and BHQ 2;

specifically, the probe for detecting the AK4 (rs 17853973) gene polymorphism provided by the invention comprises: 1 HEX-CGGGCAAGGGCACCGTGNGCCAGAGGATCGCCCG-BHQ 1;

wherein N is a variant base, specifically T > A;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-26 bp (5 ' -AAGGGCACCGTGNGCCAGAGGATC-3 '), two reverse complementary stem sequences are arranged on two sides of the loop sequence, and the stem sequence is (5 ' -CGGGC … … GCCCG-3).

The probe for detecting the AK4 (rs 12074520) gene polymorphism provided by the invention comprises the following components: 4 FAM-CCATTTTTANTTTTGAAATGG-BHQ 1;

wherein N is a variant base, specifically C > A;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-16bp (5 ' -TTANTTTTG-3 '), two reverse complementary stem sequences are arranged on two sides of the loop sequence, and the stem sequence is (5 ' -CCATTT … … AAATGG-3).

The probe for detecting the AK4 (rs 4915685) gene polymorphism provided by the invention comprises the following components: 7 CY 5-CCTGGTGAACCNTTAGTCCAGG-BHQ 2;

wherein N is a variant base, specifically G > A;

the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-18bp (5 ' -TGAACCNTTAGT-3 '), two reverse complementary stem sequences are arranged on two sides of the loop sequence, and the stem sequence is (5 ' -CCTGG … … CCAGG-3).

The design principle of the probe is as follows: according to AK4 gene polymorphism rs17853973, rs12074520 and rs4915685, respectively designing probe and primer, and ensuring that the probe is in stem-loop state when DNA template does not exist at annealing temperature. The quenching group adopted by the probe is BHQ1 or BHQ2, the quenching space range is very small, and the fluorescence can be well quenched only when the molecular beacon is in a stem-loop structure by matching with a fluorescent group with short emission wavelength, such as HEX. When the loop sequence is complementary with the target DNA sequence, the optimal PCR primer is determined to be 40-45bp in size, and the length of the PCR product is 100-150 bp.

The invention also provides 3 groups of primers for detecting AK4 gene polymorphism, the sequences of the primers are shown in SEQ ID NO. 2, 5 and 8 (primer 1) and SEQ ID NO. 3, 6 and 9 (primer 2):

AK4（rs17853973）

SEQ ID NO.:2 5′- AGGTGACACTATAGAATAATCAATGGCTTCCAAACTCCTG-3′

SEQ ID NO.:3 5′- GCAAGCCCTCACGTAGCGAAATGTTCTCCCGCAAGAAGTG -3′。

AK4（rs12074520）

SEQ ID NO.:5 5′- AGGTGACACTATAGAATAATCAATGTGTCATCTAGTATTTTCCAA-3′

SEQ ID NO.:6 5′-GCAAGCCCTCACGTAGCGAAGGATAGGCATGAAACTTTCCAC-3′。

AK4（rs4915685）

SEQ ID NO.:8 5′- AGGTGACACTATAGAATAATGAAATGGCCCAACTGTTGTC -3′

SEQ ID NO.:9 5′- GCAAGCCCTCACGTAGCGAAACTGTCTTAGCCTGGCAGCA-3′。

the invention also provides a kit for detecting AK4 gene polymorphism, which comprises the probe and/or primer;

the kit also comprises Taq enzyme, dNTP, and/or Mg2+, and instructions for use.

The invention also provides a method for detecting the AK4 gene polymorphism, which comprises the following steps:

(1) collecting a sample and extracting DNA;

(2) carrying out fluorescent quantitative PCR reaction by using the probe and the primer;

(3) and (3) analyzing the result by using a PCR instrument matched software, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks.

Wherein the total PCR reaction system is 15ul (comprising 7.5ul of PCR Mix, 0.5ul of each forward primer solution in three groups, 0.5ul of each reverse primer solution in three groups, 0.1ul of each probe in 3 groups, 2ul of sample DNA and 2.2ul of sterilized double distilled water); carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 92-97 ℃ for 5-15 minutes; denaturation at 92-97 deg.C for 10-30 s, annealing at 57-65 deg.C for 10-30 s, extension at 70-75 deg.C for 10-30 s, and 40-50 cycles; extension at 72 ℃ for 10 min; denaturation at 92-97 deg.C for 1 min, renaturation at 40 deg.C for 1 min, and real-time monitoring of fluorescence signal at 45-80 deg.C, and recording 5 times at 1 deg.C per time.

The kit can detect the polymorphism of the AK4 gene, can realize the rapid screening of the polymorphism of the AK4 gene, and can research the genotype and phenotype (such as exercise performance, energy metabolism and the like) of a large sample amount.

Although defects in AKs are known to cause some specific diseases, little is known about complex diseases caused by low AK activity, such as degenerative diseases. ATP is the most important molecule carrying high energy, and when cells play different functions, the amount of ATP produced is different, and the whole energy deficiency can cause various organ failures. By researching energy metabolism related enzymes and better understanding of a phosphate group transfer mechanism, a solid theoretical basis can be provided for the future development and treatment of diseases caused by energy metabolism disorder.

The genetic polymorphism forms the genetic basis of different individual susceptibility to diseases and different responses to drugs and environmental factors, and the selection of the appropriate genetic polymorphism is very important for understanding the nature of the phenotype on the genetic level.

The invention utilizes a fluorescent probe capable of specifically identifying a nucleic acid sequence, releases fluorescent dye through conformational change after hybridization with a target sequence, and judges a typing result according to peak patterns at different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected. Compared with other genetic typing techniques, the method is simple to operate, 96 cases of detection can be completed within 2-3 hours, the method has the advantages of high sensitivity, good specificity, rapidness, high-flux detection and the like, the fluorescence labeling probe can use different fluorophores at different sites, multiple detections can be realized, the detection result can be obtained by directly detecting the fluorescence signal in the PCR process, the genotyping is clear, the PCR post-treatment or the electrophoresis detection is not needed, and the real closed-tube operation can be realized.

Drawings

Three peak types (TT type, AA type and AT type) of the rs17853973 site in the picture 1;

fig. 2, three peak types (CC type, AA type, AC type) at the rs12074520 locus;

FIG. 3, three peak types (GG type, AA type, AG type) at rs4915685 site;

FIG. 4 is a graph showing melting curves of multiple samples.

Detailed Description

Example 1: probe and primer design

The invention designs probes and primer sequences aiming at three SNP sites of AK4 gene. The specific principle is that the fluorescent probe and a target sequence are hybridized to release fluorescent dye through conformational change, and a genotyping result is judged according to peak graphs and Tm values of different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected.

And designing a probe and a primer to realize that the probe is in a stem-loop state when the template does not exist at the annealing temperature. Taking AK4 rs17853973 as an example, the sequence comprises a loop sequence and a stem sequence with two sides in reverse complementary, the total length is 34bp, wherein the loop sequence is 24bp (5 '-AAGGGCACCGTGNGCCAGAGGATC-3'), and the stem sequence is formed by 5 bases at two ends; and the stem sequences at the two ends are just complementary; the adopted quenching group is BHQ1 matched with a fluorescent group HEX with short emission wavelength. The loop sequence is complementary with the target DNA sequence, the size of the PCR primer is determined to be 38-40bp, and the length of the PCR product is 133 bp.

The sequences of the primers and probes for AK4 (rs 17853973) are as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATAATCAATGGCTTCCAAACTCCTG-3', respectively;

sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAAATGTTCTCCCGCAAGAAGTG-3', respectively;

sequence of the probe: 5 '-HEX-CGGGCAAGGGCACCGTGNGCCAGAGGATCGCCCG-3' BHQ 1.

Wherein N is a variant base, specifically T > A;

the AK4 (rs 12074520) primer and probe sequences were as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATAATCAATGTGTCATCTAGTATTTTCCAA-3', respectively;

sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAAGGATAGGCATGAAACTTTCCAC-3', respectively;

sequence of the probe: 5 '-FAM-CCATTTTTANTTTTGAAATGG-3' BHQ 1.

Wherein N is a variant base, specifically C > A

The sequences of the primers and probes for AK4 (rs 4915685) are as follows:

sequence of primer 1: 5'-AGGTGACACTATAGAATAATGAAATGGCCCAACTGTTGTC-3'

Sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAAACTGTCTTAGCCTGGCAGCA-3', respectively;

sequence of the probe: 5 '-CY 5-CCTGGTGAACCNTTAGTCCAGG-3' BHQ 2.

Wherein N is a variant base, specifically G > A.

The above-mentioned probe and primer were synthesized by Biotechnology engineering (Shanghai) Ltd.

Example 2: standard substance for detecting different genotypes

1. The plasmid is used for constructing and preparing a wild type standard plasmid and a mutant type standard plasmid (plasmid source and the synthesis of the plasmid containing the target gene is synthesized by the company Limited in Biotechnology engineering (Shanghai) for synthesis of the plasmid containing the target gene AK4 rs17853973 and rs4915685 sites, the accuracy of the sequence is determined by sanger sequencing, the wild type standard plasmid rs17853973 genotype is TT, the rs12074520 genotype is CC, and the rs4915685 genotype is GG, the mutant type standard plasmid rs17853973 genotype is AA, the rs12074520 genotype is AA, and the rs4915685 genotype is AA, and the concentration of the standard plasmid DNA is normalized to 10 ng/ul.

2. The probe and primer in example 1 were used.

3. And (3) PCR reaction system:

1) sequentially adding 7.5ul of PCR Mix, 0.5uM of 3 groups of forward primer solutions, 0.5uM of 3 groups of reverse primer solutions and 0.1uM of 3 groups of probes into each PCR reaction hole, then respectively adding 2ul of wild type standard plasmid DNA, mutant type standard plasmid DNA and mixed type DNA (the wild type standard plasmid and the mutant type standard plasmid are mixed according to a ratio of 1: 1) into 3 different PCR reaction holes, and supplementing 15ul of sterile redistilled water;

2) carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.

4. And (3) analyzing results by using a PCR instrument matched software SLAN, and displaying different genotypes based on the difference of Tm values displayed by hybridization peaks formed by different channels. The peak AT the rs17853973 site appears AT 58 ℃ and is the TT genotype, the peak appears AT 63 ℃ and is the AT genotype (figure 1). The peak at the rs12074520 locus appeared at 54 ℃ and was of AA genotype, at 59 ℃ and was of CC genotype, and the peaks at both positions were of AC genotype (FIG. 2). The peak at rs4915685 locus appeared at 55 deg.C and was the GG genotype, the peak appeared at 48 deg.C and was the AA genotype, and the peaks at both positions were the AG genotype (FIG. 3).

Example 3: double-blind experimental investigation using AK4 gene polymorphism detection kit

1. Extracting the genome DNA of oral epithelial cells of 100 healthy volunteers in Shanghai region by a silica gel adsorption method, detecting the concentration and purity of the DNA by an electrophoresis gel imaging method, and marking the concentration of the DNA of a sample to be detected to 10 ng/ul.

2. The detection method comprises the following steps: sequentially adding 7.5ul of PCR Mix, 0.5uM of forward primer solution and 0.5uM of reverse primer solution into each PCR reaction hole, 0.1uM of probe, simultaneously detecting weak positive control (AT for rs17853973 genotype, AC for rs12074520 genotype, AG for rs4915685 genotype), negative control (TT for rs17853973 genotype, CC for rs12074520 genotype, GG for rs4915685 genotype) and a sample to be detected, adding 2ul of DNA into each reaction hole, and supplementing 15ul of sterile redistilled water; carrying out reaction on a fluorescent quantitative PCR detector, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.

3. And analyzing the result by using matched software, and displaying different genotypes based on the difference of Tm values displayed by hybridization peaks formed by different channels. The peak AT the rs17853973 site appears AT 58 ℃ and is the TT genotype, the peak appears AT 63 ℃ and is the AT genotype (figure 1). The peak at the rs12074520 locus appeared at 54 ℃ and was of AA genotype, at 59 ℃ and was of CC genotype, and the peaks at both positions were of AC genotype (FIG. 2). The peak at rs4915685 locus appeared at 55 deg.C and was the GG genotype, the peak appeared at 48 deg.C and was the AA genotype, and the peaks at both positions were the AG genotype (FIG. 3).

As shown in fig. 4, the melting curve of the multi-sample is shown, and the success rate of typing reaches 100%.

4. The genomic DNA of 100 cases of oral epithelial cells is subjected to sanger sequencing at the same time, and the detection result completely accords with the detection result of the invention, so that the accuracy of the polymorphism result of the AK4 gene applied by the method is high.

Sequence listing

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