CN111593104A - Artificial simulation nucleic acid molecular beacon and kit for detecting polymorphism of rs2108622 site of CYP4F2 gene - Google Patents

Artificial simulation nucleic acid molecular beacon and kit for detecting polymorphism of rs2108622 site of CYP4F2 gene Download PDF

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CN111593104A
CN111593104A CN201910123122.2A CN201910123122A CN111593104A CN 111593104 A CN111593104 A CN 111593104A CN 201910123122 A CN201910123122 A CN 201910123122A CN 111593104 A CN111593104 A CN 111593104A
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molecular beacon
sequence
kit
cyp4f2 gene
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葛猛
潘世让
余倩
杜柏均
王宏伟
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a typing detection method and a kit for CYP4F2 gene rs2108622 site polymorphism. The invention adopts CYP4F2 gene specific primers SEQ1 and SEQ2 to amplify CYP4F2 gene fragments, and designs CYP4F2 gene specific artificial simulated nucleic acid molecular beacons SEQ3-FAM and SEQ4-VIC in an amplification region defined by the CYP4F2 gene specific primers. The method for judging the polymorphism of the rs2108622 locus of the CYP4F2 gene based on the gene specificity PCR combined with the artificial simulated nucleic acid molecular beacon, which is 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 simulation nucleic acid molecular beacon and kit for detecting polymorphism of rs2108622 site of CYP4F2 gene
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a typing detection method and a kit for CYP4F2 gene rs2108622 site polymorphism.
Background
Thrombi, commonly colloquially referred to as clots, are the final product of the hemostatic clotting step. Thrombi consist of insoluble fibrin, deposited platelets, accumulated leukocytes and entrapped erythrocytes. When the organism is injured, the thrombus has the function of stopping bleeding; but will cause damage to the body when a thrombus obstructs blood flow through a healthy blood vessel, causing partial or complete blockage of the blood vessel. Thromboembolic diseases are pathological processes in which a thrombus is detached from a formation site, and some blood vessels are partially or completely blocked in the process of moving along with blood flow, so that ischemia, anoxia, necrosis (arterial thrombosis) and blood stasis and edema (venous thrombosis) of corresponding tissues and/or organs are caused.
Warfarin is one of coumarin anticoagulants and has an anti-vitamin K effect in vivo. Can inhibit the synthesis of blood coagulation factors II, VII, IX and X in liver, and is mainly used for preventing and treating thromboembolic diseases. As the most commonly used oral anticoagulants, the therapeutic window is rather narrow and the dosage must be adjusted according to the International Normalized Ratio (INR). Excessive use of the composition may result in a high risk of bleeding, and excessive use of the composition may result in embolism. Many clinical and environmental factors, as well as genetic mutations, affect warfarin dosage requirements.
Cytochrome P4504F 2 (CYP 4F 2) is a member of the cytochrome P450 superfamily, a gene that is part of the cytochrome P450 gene cluster on chromosome 19. Cytochrome P450 is a monooxygenase that catalyzes many reactions involved in drug metabolism, cholesterol, steroids, and other lipid synthesis. The protein is localized in endoplasmic reticulum, and starts the inactivation and degradation process of leukotriene B4 which is a potent inflammatory mediator. The rs2108622 (C/T) site polymorphism in the gene locus is closely related to the dosage requirement of warfarin, and carriers of CT and TT genotypes need higher warfarin dosage than CC genotypes, for example, in Chinese Han population, the average daily warfarin dosage of CT type and TT type patients is 7% and 18% higher than that of CC type respectively. Therefore, the site should be tested prior to prescribing to determine the optimal dosage to be administered.
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 the polymorphic site rs2108622 of CYP4F2 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 the rs2108622 site polymorphism of the human CYP4F2 gene.
The molecular beacon for detecting the polymorphism of the rs2108622 locus of the human CYP4F2 gene provided by the invention 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 are both targeted to the rs2108622 locus of the CYP4F2 gene. Wherein the molecular beacon A targets the 'C' of the locus rs2108622 of the CYP4F2 gene; the molecular beacon B targets the 'T' of the rs2108622 locus of the CYP4F2 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 rs2108622 site polymorphism of the human CYP4F2 gene.
The reagent set for detecting the polymorphism of the rs2108622 locus of the human CYP4F2 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 rs2108622 site polymorphism of the human CYP4F2 gene.
The kit for detecting the polymorphism of the rs2108622 locus of the human CYP4F2 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, etcAnd (3) a reagent. 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 site rs2108622 of a CYP4F2 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 site rs2108622 of a CYP4F2 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 the rs2108622 site polymorphism of the human CYP4F2 gene.
The invention also provides application of the molecular beacon or the kit in predicting or assisting in predicting warfarin administration dosage of a patient to be tested.
In order to achieve the above object, the present invention finally provides a method for detecting the rs2108622 site polymorphism of the human CYP4F2 gene.
The method for detecting the polymorphism of the rs2108622 locus of the human CYP4F2 gene comprises the following steps: and detecting a sample to be detected by using the molecular beacon or the reagent set, and determining the rs2108622 site polymorphism of the CYP4F2 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 polymorphism of the locus rs2108622 of the CYP4F2 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 site rs2108622 of the CYP4F2 gene of the sample to be detected is or is a candidate for 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 site rs2108622 of the CYP4F2 gene of the sample to be detected is or is a candidate of the genotype TT;
and if the sample to be detected releases the VIC fluorescent signal and the FAM fluorescent signal, and the FAM fluorescent signal value and the VIC fluorescent signal value are both continuously increased, the genotype of the site rs2108622 of the CYP4F2 gene of the sample to be detected is or is selected as the CT genotype.
The CC genotype refers to a homozygote of the bases of the rs2108622 locus of the CYP4F2 gene on two homologous chromosomes of the DNA of the sample to be detected, which are C;
the TT genotype is a homozygote of the bases of the rs2108622 locus of the CYP4F2 gene on two homologous chromosomes of the DNA of the sample to be detected, which are T;
the CT genotype refers to a heterozygote of the base of the rs2108622 locus of the CYP4F2 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 an embolic patient to be tested.
In the above molecular beacon or the kit of parts or the application or the method, the locus rs2108622 of the CYP4F2 gene is located at the 51 st position of the sequence 1.
Compared with the prior art, the invention has the following beneficial effects: the method for judging the polymorphism of the rs2108622 locus of the CYP4F2 gene based on the gene specificity PCR combined with the artificial simulated nucleic acid molecular beacon, which is 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 unnatural nucleotide isoguanine nucleotide residue (isoG) and the unnatural nucleotide 5-methylisocytosineDeoxynucleotide 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 CC genotype specific amplification curve of site rs2108622 of the human CYP4F2 gene in example 2 of the invention.
FIG. 5 is a schematic diagram of the TT genotype specific amplification curve of the human CYP4F2 gene rs2108622 site in example 2 of the invention.
FIG. 6 is a schematic diagram of a human CYP4F2 gene rs2108622 site CT genotype-specific amplification curve in example 2 of the 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
gaatggacaa aaacagagag aggggccccg cacctcaggg tccggccaca yagctgggtt 60
gtgatgggtt ccgaaaacac tgatgaggca gataatgcct g 101
<210>2
<211>31
<212>DNA
<213> Artificial sequence
<400>2
ccgacaccgg ccacacagct gggtttgtcg g 31
<210>3
<211>31
<212>DNA
<213> Artificial sequence
<400>3
ccgacaccgg ccacatagct gggtttgtcg g 31
<210>4
<211>23
<212>DNA
<213> Artificial sequence
<400>4
gaatggacaa aaacagagag agg 23
<210>5
<211>21
<212>DNA
<213> Artificial sequence
<400>5
caggcattat ctgcctcatc a 21
<210>6
<211>19
<212>DNA
<213> Artificial sequence
<400>6
ccggccacac agctgggtt 19
<210>7
<211>19
<212>DNA
<213> Artificial sequence
<400>7
ccggccacat agctgggtt 19

Claims (8)

1. The molecular beacon for detecting the rs2108622 site polymorphism of the human CYP4F2 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 polymorphism of the rs2108622 locus of the human CYP4F2 gene, which comprises the molecular beacon of any one of claims 1 to 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 to 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 rs2108622 site polymorphism of the human CYP4F2 gene, comprising the molecular beacon of any one of claims 1 to 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 polymorphism at the rs2108622 locus of the human CYP4F2 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 warfarin dosing in a patient to be tested.
8. The method for detecting the rs2108622 site polymorphism of the human CYP4F2 gene comprises 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 the rs2108622 site polymorphism of the CYP4F2 gene in the sample to be detected according to the change of a fluorescence signal in the sample to be detected.
CN201910123122.2A 2019-02-20 2019-02-20 Artificial simulation nucleic acid molecular beacon and kit for detecting polymorphism of rs2108622 site of CYP4F2 gene Pending CN111593104A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690888A (en) * 2012-06-15 2012-09-26 向华 Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system
CN109355377A (en) * 2018-12-06 2019-02-19 厦门大学 Gene associated with individualized medication of warfarin polymorphic detection kit and the preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690888A (en) * 2012-06-15 2012-09-26 向华 Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system
CN109355377A (en) * 2018-12-06 2019-02-19 厦门大学 Gene associated with individualized medication of warfarin polymorphic detection kit and the preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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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