CN111593106A - Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene - Google Patents
Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene Download PDFInfo
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Abstract
The invention discloses a typing detection method and a kit for rs12041331 site polymorphism of a PEAR1 gene. The invention adopts PEAR1 gene specific primers SEQ1 and SEQ2 to amplify a PEAR1 gene fragment, and simultaneously designs PEAR1 gene specific artificial simulated nucleic acid molecular beacons SEQ3-FAM and SEQ4-VIC in an amplification region defined by the PEAR1 gene specific primers. The method for judging the rs12041331 locus polymorphism of the PEAR1 gene based on the gene specificity PCR combined with the artificial simulated nucleic acid molecular beacon, provided by the invention, has the advantages of high accuracy, high detection speed, simplicity in operation, objective result interpretation, less closed-tube reaction pollution and the like, and is very suitable for large-scale clinical development.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a typing detection method and a kit for rs12041331 site polymorphism of a PEAR1 gene.
Background
Acute Coronary Syndrome (ACS) is a group of clinical syndromes in which the coronary artery blood flow is reduced due to the generation of thrombus resulting from the rupture or invasion of the atherosclerotic plaque of the coronary artery. As a result of reduced coronary blood flow, portions of the heart muscle fail to function properly or die. The most common symptom is chest pain, often spreading to the left shoulder or angle of the mandible, with nausea and sweating. Many ACS patients experience symptoms other than chest pain, particularly in women, elderly patients, and diabetics.
Dual antiplatelet therapy with aspirin and clopidogrel is currently the most commonly used regimen for treating patients with ACS. Although this regimen works in most patients, there are still significant individual differences. Existing results indicate that the therapeutic response of clopidogrel and aspirin is affected by genetic variation. Wherein platelet endothelial cell aggregation receptor 1 (PEAR 1) is a gene associated with aspirin response. It encodes a type 1 membrane protein, is involved in megakaryocyte and platelet formation by enhancing platelet α IIb β 3 activation, and stabilizes platelet aggregation. Among the genetic variations of PEAR1, rs12041331 (A/G) may alter its expression. Aspirin-epinephrine-induced platelet aggregation was significantly reduced in the a carriers compared to the G carriers, and AA-induced platelet aggregation was not affected by the aspirin dose. Therefore, the detection of the rs12041331 (A/G) site polymorphism can help to provide a reasonable medication scheme for different ACS patients.
At present, methods for detecting gene polymorphism mainly include a PCR-Sanger sequencing method, a chip hybridization method, a high-resolution melting curve method and the like. Although these methods can detect gene polymorphisms to some extent, they have considerable limitations. The Sanger sequencing method has more steps, needs PCR post-treatment, is complex to operate, is easy to cause pollution, and cannot meet clinical requirements. The chip hybridization method is complicated in operation, and detection thereof depends on expensive equipment and instruments, resulting in high cost. The high-resolution dissolution curve method has high requirements on instruments, can be used only by a machine which is provided with high-resolution software and is sensitive to temperature, and has difficulty in clinical popularization. The fluorescent quantitative PCR based on the Taqman hydrolysis probe cuts off the probe to generate a fluorescent signal by utilizing the exonuclease activity of Taq enzyme, and the fluorescent quenching is not thorough due to the fact that a fluorescent group and a quenching group of the Taqman probe are not close to each other closely, and a background fluorescent signal exists. In addition, the Taqman probe has poor single base mismatch recognition capability, easily generates a non-specific fluorescent signal, interferes result interpretation, and further influences the detection accuracy. Therefore, a simple, convenient, high-sensitivity, accurate and reliable method for detecting gene polymorphism is urgently needed clinically.
The Molecular Beacon (Molecular Beacon) is in a hairpin type in spatial structure and consists of a circular region and a stem region, wherein the circular region is complementary with a target DNA sequence and is about 15-35 nucleotides long, the stem region is about 5-7 nucleotides long, the stem region is formed by a complementary sequence which has higher GC content and is irrelevant with the target sequence, and the 5 'end of the Molecular Beacon is marked with a fluorescent group (F) and the 3' end of the Molecular Beacon is marked with a quenching group (Q). In the case of molecular beacons, the fluorescent group is close to the quencher group (about 7-10 nm) in the free state. At the moment, fluorescence resonance energy transfer occurs, so that fluorescence emitted by the fluorescent group is absorbed by the quenching group and emitted in a thermal form, the fluorescence is almost completely quenched, and the fluorescence background is extremely low. When the circular region of the molecular beacon is hybridized with target DNA with completely complementary sequence to form a double-stranded hybrid, the stem region of the molecular beacon is pulled apart, and the distance between the fluorescent group and the quenching group is increased. According to Foerster's theory, the efficiency of central fluorescence energy transfer is inversely proportional to the 6 th power of the distance between the two, and therefore, the fluorescence of the molecular beacon is almost 100% recovered after hybridization, and the detected fluorescence intensity is proportional to the amount of target DNA in solution (FIG. 1). Thus, the ideal molecular beacon is more efficient than the Taqman hydrolysis probe. However, the introduction of a stem region in the molecular beacon, which is not related to the target sequence, often results in some non-specific interaction between the molecular beacon and the template sequence, which leads to an increase in background signal, and thus, affects the detection efficiency. To eliminate this background signal, high requirements are imposed on the design of the molecular beacon, especially on the sequence design of the stem region. In addition, studies have shown that molecular beacons have a good effect for detecting gene mutations (including single-base mismatches, deletions, or insertion mutations) when the sequence of the loop region is short, but in practice, in many cases, the sequence of the loop region is too long due to the low GC content of a specific target sequence region, thereby affecting the detection efficiency. Therefore, it is often difficult to obtain an ideal molecular beacon.
The development of modifications to bases, i.e.the development of artificially simulated non-natural nucleotide pairs, has been known for nearly 40 years, in which heterocytes are presentPyrimidine deoxynucleotide-isoguanine deoxynucleotide (isoC-isoG) and its derivative 5-methyl isocytosine deoxynucleotide-isoguanine deoxynucleotide (iso)MeC-isoG) is classical. The work on the nucleotide pairs in isoC-isoG was first carried out by the American famous synthetic biologist Benner SA, whose team realized the entire central principle of replication, transcription and even translation of isocytosine deoxynucleotide-isoguanine deoxynucleotide (isoC-isoG) artificial expanded nucleic acids in vitro. As shown in FIG. 2, isoC and isoG are isomers of natural nucleotides C and G, respectively, which can perfectly pair themselves but cannot form a pair with natural nucleotides.
In addition to the above manual modification of base structure, there is a large class of non-natural nucleic acids based on modification of base sugar rings, such as Locked Nucleic Acids (LNA). LNA, which broadly refers to an oligonucleotide sequence containing one or more LNA monomers (locked nucleotides), is an artificial mimic nucleic acid that has been rapidly developed in recent years and has been widely used in the fields of molecular diagnostics, gene therapy, and the like. As shown in fig. 3, a methylene bridge is formed between the 2 '-O and 4' -C of the pentose ring of the LNA monomer. LNA does not alter the base pairing of natural nucleic acids, but has greater affinity and greater mismatch recognition relative to natural nucleic acids.
Disclosure of Invention
The invention aims to provide a novel PEAR1 gene rs12041331 polymorphic site typing detection method and a kit based on a molecular beacon of artificial simulated nucleic acid.
In order to achieve the purpose, the invention firstly provides a molecular beacon for detecting the rs12041331 site polymorphism of the human PEAR1 gene.
The molecular beacon for detecting the rs12041331 locus polymorphism of the human PEAR1 gene consists of a molecular beacon A and a molecular beacon B;
the sequence of the molecular beacon A is a sequence 2 in a sequence table, wherein the 2 nd position of the sequence 2 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues;
the sequence of the molecular beacon B is a sequence 3 in a sequence table, wherein the 2 nd position of the sequence 3 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues.
The 7 th to 25 th sites of the molecular beacon A and the molecular beacon B are both circular region sequences, and the 1 st to 6 th sites and the 26 th to 31 th sites are both stem region sequences.
The ring regions of the molecular beacon A and the molecular beacon B target the locus rs12041331 of the PEAR1 gene. Wherein the molecular beacon A targets the 'A' of the locus rs12041331 of the PEAR1 gene; the molecular beacon B targets the 'G' of the locus rs12041331 of the PEAR1 gene.
Furthermore, two ends of the molecular beacon A and the molecular beacon B are also marked with a fluorescent group and a quenching group, and the fluorescent groups marked by the molecular beacon A and the molecular beacon B are different. The molecular beacon A and the molecular beacon B can be the same or different in labeled quenching group.
In each molecular beacon, the fluorescence emitted by the fluorophore can be absorbed by the quencher. The fluorescent group and the quenching group can be respectively positioned at the 5 'terminal and the 3' terminal of the basic molecular beacon, and the positions of the fluorescent group and the quenching group can be exchanged as long as the requirement that the fluorescence emitted by the fluorescent group in the basic molecular beacon in a free state can be quenched by the quenching group is met.
Further, the fluorophore may be FAM, Hex, TET, Cy3, JOE; the quencher group can be Dabcyl, TAMRA. In the invention, the 5 'end of the molecular beacon A is marked with FAM fluorescent group, and the 3' end is marked with Dabcyl quenching group; the 5 'end of the molecular beacon B is marked with a VIC fluorescent group, and the 3' end is marked with a Dabcyl quenching group.
In order to achieve the purpose, the invention further provides a kit for detecting the rs12041331 site polymorphism of the human PEAR1 gene.
The reagent set for detecting the rs12041331 site polymorphism of the human PEAR1 gene consists of the molecular beacon and a primer pair which can be amplified from a human genome and contains the recognition sequence of the circular region of the molecular beacon.
In the above-mentioned kit, the primer pair is composed of a single-stranded DNA represented by sequence 4 in the sequence table and a single-stranded DNA represented by sequence 5 in the sequence table.
In the above kit, the molecular beacon and the primer pair are packaged independently. The molar ratio of the molecular beacon A to the molecular beacon B in the molecular beacon can be 1: 1; the molar ratio of the two single-stranded DNAs in the primer pair may be 1: 1. The molar ratio of the molecular beacon A and the molecular beacon B in the kit to the two single-stranded DNAs of the primer pair can be 2:2:5: 5.
In order to achieve the purpose, the invention also provides a kit for detecting the rs12041331 site polymorphism of the human PEAR1 gene.
The kit for detecting the rs12041331 site polymorphism of the human PEAR1 gene comprises the molecular beacon or the reagent set.
The kit can also comprise positive quality control, negative quality control and other reagents. The other reagents can be reaction buffer, dNTPs and MgCl2Solution, DNA polymerase and/or nuclease-free water. The positive quality control comprises a recombinant plasmid 1, a recombinant plasmid 2 and a recombinant plasmid 3. The recombinant plasmid 1 is obtained by replacing a DNA fragment between EcoRV and SmaI recognition sequences in an escherichia coli cloning vector pUC57 with a DNA fragment shown in a sequence 1 (the locus rs12041331 of a PEAR1 gene in the sequence 1 is A); the recombinant plasmid 2 is obtained by replacing a DNA fragment between EcoRV and SmaI recognition sequences in an escherichia coli cloning vector pUC57 with a DNA fragment shown in a sequence 1 (the locus rs12041331 of a PEAR1 gene in the sequence 1 is G); the recombinant plasmid 3 is the recombinant plasmid 1 andthe recombinant plasmid 2 is obtained by mixing according to the molar ratio of 1: 1. The negative quality control can be specifically nuclease-free water. The DNA polymerase can be EX Taq DNA polymerase.
In order to achieve the above objects, the present invention also provides a novel use of the above molecular beacon or the above kit.
The invention provides application of the molecular beacon or the reagent set in detecting human PEAR1 gene rs12041331 site polymorphism.
The invention also provides the application of the molecular beacon or the kit in predicting or assisting in predicting the treatment risk of aspirin of a patient with acute coronary syndrome.
In order to achieve the above objects, the present invention finally provides a method for detecting the rs12041331 site polymorphism of human PEAR1 gene.
The method for detecting the rs12041331 site polymorphism of the human PEAR1 gene comprises the following steps: and detecting a sample to be detected by using the molecular beacon or the reagent set, and determining the rs12041331 site polymorphism of the PEAR1 gene in the sample to be detected according to the change of a fluorescence signal in the sample to be detected.
In the method, the step of detecting the sample to be detected by using the molecular beacon or the kit of reagents is to detect the DNA of the sample to be detected by using the molecular beacon or the kit of reagents.
The method for determining the rs12041331 site polymorphism of the PEAR1 gene in the sample to be detected according to the change of the fluorescence signal in the sample to be detected comprises the following steps:
if the sample to be detected releases the FAM fluorescent signal, does not release the VIC fluorescent signal, and the value of the FAM fluorescent signal is continuously increased, the genotype of the gene rs12041331 site of the PEAR1 gene of the sample to be detected is or is selected as the AA genotype;
if the sample to be detected releases the VIC fluorescent signal, does not release the FAM fluorescent signal, and the value of the VIC fluorescent signal is continuously increased, the genotype of the gene rs12041331 site of the PEAR1 gene of the sample to be detected is or is candidate to be GG genotype;
and if the sample to be detected releases the VIC fluorescence signal and the FAM fluorescence signal, and both the FAM fluorescence signal value and the VIC fluorescence signal value are continuously increased, determining that the genotype of the PEAR1 gene rs12041331 site of the sample to be detected is the AG genotype or is a candidate.
The AA genotype refers to a homozygote of A in the basic groups of rs12041331 locus of the PEAR1 gene on two homologous chromosomes of the DNA of the sample to be detected;
the GG genotype refers to a homozygote of G at the rs12041331 site of the PEAR1 gene on two homologous chromosomes of the DNA of a sample to be detected;
the AG genotype refers to a heterozygote of A and G of bases of rs12041331 sites of PEAR1 genes on two homologous chromosomes of the DNA of a sample to be detected.
In the above method, the sample to be tested may be a blood sample of a person to be tested.
In the above molecular beacon or kit of parts or kit or use or method, the rs12041331 locus of the PEAR1 gene is located at position 51 of the sequence 1.
Compared with the prior art, the invention has the following beneficial effects: the method for judging the rs12041331 locus polymorphism of the PEAR1 gene based on the gene specificity PCR combined with the artificial simulated nucleic acid molecular beacon, provided by the invention, has the advantages of high accuracy, high detection speed, simplicity in operation, objective result interpretation, less closed-tube reaction pollution and the like, and is very suitable for large-scale clinical development.
Drawings
Fig. 1 is a schematic diagram of the operation of a molecular beacon.
FIG. 2 is a diagram of the non-natural nucleotide isoguanine nucleotide residue (isoG) and the non-natural nucleotide 5-methylisocytosine deoxynucleotide residue (iso)MeC) The structure of (1).
FIG. 3 is a diagram of the structure of locked nucleotide residues.
FIG. 4 is a schematic diagram of the AA genotype specific amplification curve at the rs12041331 locus of the human PEAR1 gene in example 2 of the present invention.
FIG. 5 is a schematic diagram of genotype-specific amplification curves of rs12041331 site GG of human PEAR1 gene in example 2 of the present invention.
FIG. 6 is a schematic diagram of the genotype-specific amplification curve of the human PEAR1 gene rs12041331 site AG in example 2 of the present invention.
FIG. 7 is a schematic diagram of the amplification curve of the standard sample 1 detected using the primer pair SEQ1 and SEQ2, the common Taqman probe SEQ5-FAM and SEQ 6-VIC.
FIG. 8 is a schematic diagram of the amplification curve of the standard sample 2 detected using the primer pair SEQ1 and SEQ2, the common Taqman probe SEQ5-FAM and SEQ 6-VIC.
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<213> Artificial sequence
<400>6
ctgtctcact tccatcaccc ttactc 26
<210>7
<211>26
<212>DNA
<213> Artificial sequence
<400>7
ctgtctcact tccgtcaccc ttactc 26
Claims (8)
1. The molecular beacon for detecting the rs12041331 site polymorphism of the human PEAR1 gene consists of a molecular beacon A and a molecular beacon B;
the sequence of the molecular beacon A is a sequence 2 in a sequence table, wherein the 2 nd position of the sequence 2 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues;
the sequence of the molecular beacon B is a sequence 3 in a sequence table, wherein the 2 nd position of the sequence 3 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues.
2. The molecular beacon of claim 1, wherein: and fluorescent groups and quenching groups are marked at two ends of the molecular beacon A and the molecular beacon B, and the fluorescent groups marked by the molecular beacon A and the molecular beacon B are different.
3. The molecular beacon of claim 2, wherein: the molecular beacon A is marked with FAM fluorophore; the molecular beacon B is marked with a VIC fluorescent group.
4. A kit for detecting the rs12041331 site polymorphism of the human PEAR1 gene, which comprises the molecular beacon of any one of claims 1-3 and a primer pair capable of amplifying the recognition sequence of the circular region of the molecular beacon of any one of claims 1-3 from the human genome.
5. The kit of claim 4, wherein: the primer pair consists of a single-stranded DNA shown in a sequence 4 in a sequence table and a single-stranded DNA shown in a sequence 5 in the sequence table.
6. A kit for detecting the rs12041331 site polymorphism of the human PEAR1 gene, comprising the molecular beacon of any one of claims 1-3 or the kit of parts of claims 4 or 5.
7. Use of the molecular beacon of any one of claims 1 to 3 or the kit of parts of claims 4 or 5 or the kit of parts of claim 6 for detecting a polymorphism at the rs12041331 site of the human PEAR1 gene;
or, the use of a molecular beacon according to any one of claims 1 to 3 or a kit of parts according to claim 4 or 5 or a kit according to claim 6 for predicting or aiding in the prediction of the risk of aspirin treatment in a patient with acute coronary syndrome.
8. A method for detecting rs12041331 site polymorphism of human PEAR1 gene, comprising the following steps: detecting a test sample by using the molecular beacon as claimed in any one of claims 1 to 3 or the kit as claimed in claim 4 or 5, and determining the rs12041331 site polymorphism of the PEAR1 gene in the test sample according to the change of the fluorescence signal in the test sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910127727.9A CN111593106A (en) | 2019-02-21 | 2019-02-21 | Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910127727.9A CN111593106A (en) | 2019-02-21 | 2019-02-21 | Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene |
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| CN111593106A true CN111593106A (en) | 2020-08-28 |
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| CN201910127727.9A Pending CN111593106A (en) | 2019-02-21 | 2019-02-21 | Artificial mimic nucleic acid molecular beacon and kit for detecting polymorphism of rs12041331 locus of PEAR1 gene |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949964A (en) * | 2018-08-21 | 2018-12-07 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of rs12041331 |
| CN109306377A (en) * | 2018-05-21 | 2019-02-05 | 上海迈浦生物科技有限公司 | SNaPShot serotype specific primer and its detection method |
-
2019
- 2019-02-21 CN CN201910127727.9A patent/CN111593106A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109306377A (en) * | 2018-05-21 | 2019-02-05 | 上海迈浦生物科技有限公司 | SNaPShot serotype specific primer and its detection method |
| CN108949964A (en) * | 2018-08-21 | 2018-12-07 | 潍坊德诺泰克生物科技有限公司 | For detecting the primed probe group and its application of rs12041331 |
Non-Patent Citations (1)
| Title |
|---|
| PINPIN SHENG 等: "Design of a novel molecular beacon: modification of the stem with artificially genetic alphabet", CHEM COMMUN (CAMB), no. 41, pages 5128 - 5130 * |
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Application publication date: 20200828 |