CN111593103A - Artificial mimic nucleic acid molecular beacon and kit for detecting rs5443 site polymorphism of GNB3 gene - Google Patents

Artificial mimic nucleic acid molecular beacon and kit for detecting rs5443 site polymorphism of GNB3 gene Download PDF

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CN111593103A
CN111593103A CN201910122797.5A CN201910122797A CN111593103A CN 111593103 A CN111593103 A CN 111593103A CN 201910122797 A CN201910122797 A CN 201910122797A CN 111593103 A CN111593103 A CN 111593103A
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molecular beacon
sequence
kit
detecting
gnb3 gene
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葛猛
潘世让
杜柏均
余倩
王宏伟
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a method and a kit for detecting rs5443 site polymorphism of a GNB3 gene by typing. The invention adopts GNB3 gene specific primers SEQ1 and SEQ2 to amplify a GNB3 gene fragment, and simultaneously designs GNB3 gene specific artificial simulated nucleic acid molecular beacons SEQ3-FAM and SEQ4-VIC in an amplification region defined by the GNB3 gene specific primer. The method for judging the rs5443 site polymorphism of the GNB3 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

Artificial mimic nucleic acid molecular beacon and kit for detecting rs5443 site polymorphism of GNB3 gene
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a typing detection method and a kit for rs5443 site polymorphism of a GNB3 gene.
Background
Major depressive disorder (MD), depression for short, is a psychiatric disorder characterized by a depressed mood in most cases over a period of at least two weeks. It is often accompanied by the phenomena of low self-esteem, loss of interest in normal life, no vitality, pain of unknown reason and the like. The patient may occasionally also have a false idea that is different from the normal. Some people do not show obvious symptoms in depression period, and some people have certain symptoms. Major depressive disorder may negatively impact an individual's life, work, education, as well as sleep, eating habits, and physical health. 2% -8% of major depressive disorder patients die from suicide, and about 50% of suicide persons suffer from depression or other mood disorders.
The incidence of major depression is 3% of the global population, with the affected population varying from 7% in japan to 21% in france. The disease is more prevalent in developed countries than in developing countries. The most common age of onset is 20-30 years, and women are affected twice as much as men.
It is now generally accepted that the development of depression is a result of a combination of genetic, environmental and psychological factors. Risk factors for disease include family history of the disease, major life changes, drug abuse and chronic health problems. Approximately 40% of the risk of disease is associated with genetic factors, and genetic-environmental interactions play an important role in the development of disease.
Guanine nucleotide binding proteins (G proteins) are key regulators of cellular responses through the cAMP pathway. Abnormal expression and function of G-protein is closely related to the pathophysiology of various psychiatric diseases, including major depression. The G protein contains multiple subunits, each encoded by several isoforms. The GNB3 gene located on chromosome 12p13.31 encodes G protein beta 3 subunit, and its functional polymorphic site rs5443 (C/T) is related to increase of signal transduction and ion transport activity, major depressive disorder risk and antidepressant therapeutic response. Individuals of the CC genotype are only sensitive to MD when exposed to high negative events, whereas CT-and TT-type individuals are sensitive to MD when exposed to low negative events. Therefore, typing the rs5443 site provides guidance for prevention and treatment of MD.
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 base-directed modification, i.e., artificial, mimetic, non-natural nucleotide pairs, studies has been in the recent 40 years, in which isocytosine deoxynucleotide-isoguanine deoxynucleotide (isoC-isoG) and its derivatives 5-methylisocytosine 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 method and a kit for detecting the typing of rs5443 polymorphic sites of a GNB3 gene 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 rs5443 site polymorphism of human GNB3 gene.
The molecular beacon for detecting the rs5443 site polymorphism of the human GNB3 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 circular regions of the molecular beacon A and the molecular beacon B are both targeted to the rs5443 locus of the GNB3 gene. Wherein the molecular beacon A targets the 'C' of the rs5443 site of the GNB3 gene; the molecular beacon B targets the 'T' at the rs5443 site of the GNB3 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 rs5443 site polymorphism of human GNB3 gene.
The kit for detecting the rs5443 site polymorphism of the human GNB3 gene provided by the invention comprises the molecular beacon and a primer pair which can be amplified from a human genome and contains a recognition sequence of a 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 rs5443 site polymorphism of human GNB3 gene.
The kit for detecting the rs5443 site polymorphism of the human GNB3 gene comprises the molecular beacon or the kit.
The kit can also comprise positive quality control, negative quality control and other reagents. The other reagents can be reaction buffer, dNTPs and MgCl2Solutions ofAnd 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 rs5443 site of a GNB3 gene in the sequence 1 is C); 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 rs5443 site of a GNB3 gene in the sequence 1 is 'T'); the recombinant plasmid 3 is obtained by mixing the recombinant plasmid 1 and the recombinant plasmid 2 according to a 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 rs5443 site polymorphism of human GNB3 gene.
The invention also provides application of the molecular beacon or the reagent set in prediction or auxiliary prediction of the risk of the major depression and the anti-depression treatment response of a person to be tested.
In order to achieve the above objects, the present invention finally provides a method for detecting rs5443 site polymorphism of human GNB3 gene.
The method for detecting the rs5443 site polymorphism of the human GNB3 gene comprises the following steps: and detecting a sample to be detected by using the molecular beacon or the reagent set, and determining the rs5443 site polymorphism of the GNB3 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 rs5443 site polymorphism of the GNB3 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 rs5443 locus of the GNB3 gene of the sample to be detected is or is candidate to be the CC 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 rs5443 locus of the GNB3 gene of the sample to be detected is the TT genotype or is a candidate;
and if the sample to be detected releases the VIC fluorescence signal and the FAM fluorescence signal, and the FAM fluorescence signal value and the VIC fluorescence signal value are both continuously increased, determining that the genotype of the site rs5443 of the GNB3 gene of the sample to be detected is or is candidate to be the CT genotype.
The CC genotype refers to a homozygote of C in basic groups of rs5443 locus of GNB3 gene on two homologous chromosomes of the DNA of a sample to be detected;
the TT genotype is a homozygote of T at the rs5443 locus of the GNB3 gene on two homologous chromosomes of the DNA of a sample to be detected;
the CT genotype refers to a heterozygote of the base of rs5443 site of the GNB3 gene on two homologous chromosomes of the DNA of a sample to be detected, wherein the base is C and T.
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 application or method, the rs5443 site of the GNB3 gene is located at the 51 st site of sequence 1.
Compared with the prior art, the invention has the following beneficial effects: the method for judging the rs5443 site polymorphism of the GNB3 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 a non-natural nucleotide isoguanine nucleotide residue (isoG) and a non-natural nucleotide 5-methylisocytosine deoxynucleotide residueRadical (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 a CC genotype-specific amplification curve of the rs5443 site of the human GNB3 gene in example 2 of the present invention.
FIG. 5 is a schematic diagram of TT genotype-specific amplification curves of the rs5443 site of the human GNB3 gene in example 2 of the present invention.
FIG. 6 is a schematic diagram of the CT genotype-specific amplification curve of the human GNB3 gene rs5443 site 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.
Sequence listing
<110>
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>101
<212>DNA
<213> Artificial sequence
<400>1
caggagctga tctgcttctc ccacgagagc atcatctgcg gcatcacgtc ygtggccttc 60
tccctcagtg gccgcctact attcgctggc tacgacgact t 101
<210>2
<211>31
<212>DNA
<213> Artificial sequence
<400>2
ccgacacatc acgtccgtgg ccttctgtcg g 31
<210>3
<211>31
<212>DNA
<213> Artificial sequence
<400>3
ccgacacatc acgtctgtgg ccttctgtcg g 31
<210>4
<211>21
<212>DNA
<213> Artificial sequence
<400>4
caggagctga tctgcttctc c 21
<210>5
<211>23
<212>DNA
<213> Artificial sequence
<400>5
aagtcgtcgt agccagcgaa tag 23
<210>6
<211>25
<212>DNA
<213> Artificial sequence
<400>6
cggcatcacg tccgtggcct tctcc 25
<210>7
<211>25
<212>DNA
<213> Artificial sequence
<400>7
cggcatcacg tctgtggcct tctcc 25

Claims (8)

1. The molecular beacon for detecting rs5443 site polymorphism of the human GNB3 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 rs5443 site polymorphism of human GNB3 gene, comprising the molecular beacon of any one of claims 1-3 and a primer pair capable of amplifying from human genome to obtain a primer pair containing the recognition sequence of the circular region of the molecular beacon of any one of claims 1-3.
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 rs5443 site polymorphism of human GNB3 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 the rs5443 site polymorphism of the human GNB3 gene;
or, the use of a molecular beacon as claimed in any one of claims 1 to 3 or a kit of parts as claimed in claim 4 or 5 or a kit of parts as claimed in claim 6 for predicting or aiding in the prediction of the risk of major depressive disorder and antidepressant response in a subject.
8. A method for detecting rs5443 site polymorphism of human GNB3 gene, comprising the following steps: detecting a sample to be detected 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 rs5443 site polymorphism of the GNB3 gene in the sample to be detected according to the change of a fluorescence signal in the sample to be detected.
CN201910122797.5A 2019-02-20 2019-02-20 Artificial mimic nucleic acid molecular beacon and kit for detecting rs5443 site polymorphism of GNB3 gene Pending CN111593103A (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101240320A (en) * 2007-02-06 2008-08-13 上海主健生物工程有限公司 Kit for detecting cardiovascular disease incidence inheritance risk
CN101354343A (en) * 2007-07-27 2009-01-28 上海主健生物工程有限公司 Reagent kit for detecting adiposity genetic susceptibility
EP2463384A2 (en) * 2010-12-07 2012-06-13 Medtronic, Inc. Diagnostic kits, genetic markers and methods for SCD or SCA therapy selection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101240320A (en) * 2007-02-06 2008-08-13 上海主健生物工程有限公司 Kit for detecting cardiovascular disease incidence inheritance risk
CN101354343A (en) * 2007-07-27 2009-01-28 上海主健生物工程有限公司 Reagent kit for detecting adiposity genetic susceptibility
EP2463384A2 (en) * 2010-12-07 2012-06-13 Medtronic, Inc. Diagnostic kits, genetic markers and methods for SCD or SCA therapy selection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HU Q等: "Influence of GNB3 C825T polymorphism on the efficacy of antidepressants in the treatment of major depressive disorder: a meta-analysis", JOURNAL OF AFFECTIVE DISORDERS, vol. 172, pages 103 - 109 *
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