CN114686581A - Detection method and kit for 19bp insertion mutation of DHFR gene intron and use method of kit - Google Patents
Detection method and kit for 19bp insertion mutation of DHFR gene intron and use method of kit Download PDFInfo
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Abstract
The invention provides a method for detecting 19bp insertion mutation of a DHFR gene intron, which is characterized in that a PCR primer combination and a specific detection fluorescent probe are designed according to the 19bp of the DHFR gene intron, and real-time fluorescent quantitative PCR is carried out on a sample to be detected, so that the type of the 19bp insertion mutation of the DHFR gene intron is classified. The invention also provides application of the PCR primer combination and the specific detection fluorescent probe in preparation of a reagent for detecting the 19bp insertion mutation of the intron of the DHFR gene. The invention also provides a kit for detecting the 19bp insertion mutation of the intron of the DHFR gene.
Description
Technical Field
The invention relates to the technical field of insertion mutation detection, in particular to a detection method of a DHFR gene intron 19bp insertion mutation and a use method of a kit.
Background
Genomic DNA is converted, inverted, inserted or deleted at a specific nucleotide sequence and occurs at a frequency of 1% or more in a population, and is called a gene polymorphism. The gene polymorphism may be related to certain physiological metabolism functions and drug sensitivity, and the diagnosis at the molecular level can provide the patient with the guidance of disease prevention, individualized treatment, prognosis judgment and the like.
Dihydrofolate reductase (DHFR) in the human body uses NADPH to reduce dihydrofolate to produce tetrahydrofolate, a key enzyme in folate metabolism. The research shows that the 19bp deletion in the DHFR intron region can cause the ratio (OR) of various diseases such as spina bifida, leukemia and autism to be remarkably improved. The principle may be that the deletion of the site leads to the down-regulation of the gene expression level, thereby affecting the synthesis amount of DHFR and further affecting the physiological metabolism.
The DHFR 19bp insertion mutation detection is helpful for the clinician to formulate a corresponding prevention and treatment scheme for cardiovascular and cerebrovascular diseases. The risk of suffering from the disease can be reduced by a high-risk group through a first-level preventive measure, and patients suffering from cardiovascular and cerebrovascular diseases can be treated more specifically. The mutation site can be used for diagnosing cardiovascular and cerebrovascular diseases and evaluating the risk of high risk groups, and can also be used for guiding the use of related nutrient element supplements such as folic acid, vitamin D and the like. Even the gene can be jointly diagnosed with SNP sites of other key genes in the folic acid metabolic pathway, and a more accurate treatment scheme is provided for patients through a proper model.
For a long time, sanger sequencing method or PCR-PFLP detection technology is mostly adopted for DHFR polymorphism genotype analysis, and the PCR-PFLP technology depends on restriction endonuclease digestion and electrophoresis analysis, so that misjudgment of some results is often generated. The Sanger sequencing method has long sequencing period and low flux, needs expensive equipment and complex operation, is not suitable for large-scale screening or gives a diagnosis result quickly, and is difficult to popularize on a large scale.
Disclosure of Invention
In order to solve the problems, the application provides a method for detecting the 19bp insertion mutation of the intron of the DHFR gene, which can simultaneously detect any one of a wild type, a homozygous mutant type and a heterozygous type of the DHFR gene, design a PCR primer combination and a specific detection fluorescent probe according to the 19bp insertion mutation region of the intron of the DHFR gene, carry out real-time fluorescent quantitative PCR on a sample, and further identify any one or a combination of more of the wild type, the homozygous mutant type and the heterozygous type of the DHFR gene.
PCR primer combination:
forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3', respectively;
reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3', respectively;
specific detection of fluorescent probes:
1, probe 1: a fluorophore 5'-TCGCGCGTCCCGCCCAGGT-3' quencher group;
and (3) probe 2: fluorophore 5'-AGTCGGCCACCCCGACCGT-3' quencher.
It should be noted that the primer plays a role of a starting point for the PCR reaction, the primer is bound to the DNA template by the base complementary pairing principle in the annealing step of the PCR reaction, and the elongation reaction of base complementary pairing is performed from the 5 'end to the 3' end by Taq enzyme, so that the DNA template that is hydrogen bond broken into single strand in the denaturation step is reformed into a complete double-stranded structure, and 1 DNA molecule is changed into 2 DNA molecules.
It should be noted that the primer in the present invention refers to an oligonucleotide chain composed of a certain number of dNTPs, which is artificially synthesized by a DNA synthesizer, and can bind to a region complementary to a target nucleic acid chain to be amplified, and serve as a starting point of a PCR reaction to carry out an extension reaction of the DNA chain.
It should be noted that probe 1 is an insertion mutation sequence, and probe 2 is a sequence spanning the mutation site and not containing an insertion mutation sequence. The two probes are artificially synthesized nucleotide chains, FAM fluorescent signals and VIC fluorescent signals are respectively marked at the 5 'end, and quenching groups are marked at the 3' end. In the PCR reaction, the synthesized strand is continuously extended from the 5 'end to the 3' end by the action of Taq enzyme, and when the synthesized strand is extended to the position where the probe is bound to the template, the probe is cleaved and degraded by the 5 '→ 3' exonuclease activity of Taq enzyme. Therefore, the fluorescent group at the 5 'end of the probe is far away from the quenching group at the 3' end in space distance, the fluorescent signal is released and then captured by an instrument, and the PCR reaction can be converted into an optical signal.
Preferably, in the PCR primer combination of the detection method for the 19bp insertion mutation of the intron of the DHFR gene provided by the invention, the fluorophore used at the 5' end of the PCR probe is a fluorescence reporter group suitable for fluorescence PCR quantitative analysis, and comprises one or more of FAM, VIC, TET, HEX and ROX; the 3' quenching group of the PCR probe is one or more of BHQ-1, BHQ-2, Dabcy1 and Eclipse.
In addition, the invention also provides a kit for detecting the 19bp insertion mutation of the intron of the DHFR gene, which contains the primer combination and a specific detection fluorescent probe.
Preferably, the PCR primer combination used in the kit provided by the present invention is:
forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3', respectively;
reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3' are provided.
Preferably, the specific detection fluorescent probe applied by the kit provided by the invention is as follows:
1, probe 1: a fluorophore 5'-TCGCGCGTCCCGCCCAGGT-3' quencher group;
and (3) probe 2: fluorophore 5'-AGTCGGCCACCCCGACCGT-3' quencher.
Preferably, in the kit provided by the invention, the fluorescent group used at the 5' end of the PCR primer combination is a fluorescent reporter group suitable for fluorescent PCR quantitative analysis, and comprises one or more of FAM, VIC, TET, HEX and ROX; the 3' quenching group of the PCR primer combination is one or more of BHQ-1, BHQ-2, Dabcy1 and Eclipse.
Further, the kit provided by the invention also comprises a PCR reaction system.
Preferably, the PCR reaction system comprises: PCR buffer solution, dNTPs, Taq DNA polymerase and Mg2+One or more of specific amplification primers and deionized water.
Preferably, the invention further provides application of the PCR primer combination, the fluorescent probe and the kit thereof in detecting the 19bp insertion mutation of the intron of the DHFR gene, which comprises the following steps:
s1, extracting DNA in the sample;
s2, carrying out PCR amplification reaction by using the DNA extracted in the step S1 as a template;
and S3, analyzing and judging results.
Preferably, the PCR reaction system is:
preferably, the PCR reaction conditions are: 2min at 50 ℃; 95 ℃ for 2min,95 ℃ for 15s,60 ℃ for 1min, 50 ℃ for 1min, for a total of 45 cycles.
The beneficial effects of the invention include: the method for detecting the 19bp insertion mutation of the intron of the DHFR gene provided by the invention designs a corresponding PCR primer combination and a specific detection fluorescent probe for the first time, and develops a corresponding detection kit according to the PCR primer combination, so that whether the DHFR gene has the 19bp insertion mutation of the intron fragment can be accurately identified, a sample is divided into a wild type, a homozygous mutant type and a heterozygous mutant type, and the folate metabolism capability of the sample is further evaluated.
Drawings
FIG. 1 is an amplification curve of a wild-type sample;
FIG. 2 is an amplification curve of a homozygous mutant sample;
FIG. 3 is a heterozygous mutant sample amplification curve;
Detailed Description
The invention discloses a detection method and a kit for 19bp insertion mutation of a DHFR gene intron, and a person skilled in the art can use the content to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The materials or reagents used in the detection kit for the 19bp insertion mutation of the DHFR gene intron provided by the invention can be purchased from the market.
Example one
Discloses a PCR primer combination applied in the detection method of the 19bp insertion mutation of the DHFR gene intron and a design process of a specific detection fluorescent probe.
The specific method comprises the following steps:
firstly, acquiring a full-length reference sequence of the DHFR gene, wherein the full length of the DHFR gene is 28758bp, and a 19bp insertion mutation site of an intron of the DHFR gene is positioned between 639 → 640 bases.
Secondly, intercepting fragments with the length of 500bp respectively at the upstream and the downstream of the mutation site from the whole gene sequence of the DHFR, and using the fragments as a primer probe design region.
Thirdly, firstly searching a probe in a primer probe design region according to the following design principle:
the Tm value is usually 65-72 ℃;
② the 5 'end can not be G base, because even if the probe is cut by enzyme, the 5' end G base still has the function of quenching the report fluorescence;
thirdly, single nucleotide is prevented from forming strings;
fourthly, when the probe is annealed, the probe is close to the primer as much as possible and can not be overlapped, and the distance from the 3' end of the primer is at least 1 base;
according to the design principle, two probe sequences are preferably selected in the invention:
1, probe 1: a fluorophore 5'-TCGCGCGTCCCGCCCAGGT-3' quenching group which is a fluorophore,
and (3) probe 2: fluorophore 5'-AGTCGGCCACCCCGACCGT-3' quencher.
Probe 1 is an insertion mutation sequence, and probe 2 is a sequence which spans the mutation site and does not contain the insertion mutation sequence. The two probes are artificially synthesized nucleotide chains, FAM fluorescent signals and VIC fluorescent signals are respectively marked at the 5 'end, and quenching groups are marked at the 3' end. During the PCR reaction, the synthesized strand is continuously extended from the 5 'end to the 3' end by the action of Taq enzyme, and when the synthesized strand is extended to the position where the probe binds to the template, the probe is cleaved and degraded by the 5 '→ 3' exonuclease activity of Taq enzyme. Therefore, the fluorescent group at the 5 'end of the probe is far away from the quenching group at the 3' end in space distance, the fluorescent signal is released and then captured by an instrument, and the PCR reaction can be converted into an optical signal.
After the probe sequence is determined, designing a PCR primer combination according to the following design principle:
firstly, the length of a general primer is 15-25 bp;
the G/C content is 20-80%;
the Tm value of the primer is generally 5-10 ℃ less than that of the probe; (ii) a
The Tm values of a pair of primers are generally not suitable to be different greatly;
fifthly, the PCR fragment is not suitable to be overlong, and is generally about 70-500 bp;
and sixthly, excessive secondary structure is avoided as much as possible.
According to the design principle of the PCR primer combination, a plurality of pairs of primers which are relatively in accordance with the design principle are found from the primer probe design region. By matching different primer pairs with the probes in the above and then performing performance tests, the sequence of the PCR primer combination is preferably as follows:
forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3'
Reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3'
The primer pair plays a role of a starting point for PCR reaction, the primer is combined on a DNA template in the annealing step of the PCR reaction according to the base complementary pairing principle, and the extension reaction of base complementary pairing is carried out from the 5 'end to the 3' end through Taq enzyme, so that the DNA template which is changed into a single strand by hydrogen bond breakage in the denaturation step is reformed into a complete double-stranded structure, and 1 DNA molecule is changed into 2 DNA molecules.
The primer in the present invention refers to an oligonucleotide chain composed of a certain number of dNTPs, which is synthesized by a DNA synthesizer, and can bind to a region complementary to a target nucleic acid chain to be amplified, and serve as a starting point of a PCR reaction to perform an extension reaction of the DNA chain.
Example two
The embodiment provides a method for detecting a 19bp insertion detection mutation of a DHFR gene intron in a sample, which comprises the following steps:
a. and (3) extracting DNA, namely extracting the DNA of a blood sample or the DNA of an oral swab sample by using a nucleic acid extraction reagent in the tyler medical production, wherein the specific operation is shown in the specification. Storing at-20 deg.C for use.
b. And (3) PCR reaction: taking the DNA solution as a template, carrying out PCR reaction by using a PCR reaction system of the kit, and amplifying to obtain a PCR product; wherein, the PCR primer combination comprises a pair of primers of PCR primer combination DHFR-F/DHFR-R, and the primers respectively have the following sequences:
forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3'
Reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3'
The PCR primer combination DHFR-F/DHFR-R is designed according to the DNA sequence of DHFR, and the PCR primer combination DHFR-F/DHFR-R can carry out PCR amplification on the DNA of the human genome;
the PCR reaction system is as follows:
| components | Volume of addition |
| 10×Buffer（0.25mmol/L） | 12.5μL |
| Taq enzyme (1U) | 0.5μL |
| Forward primer (0.2. mu. mol/L) | 0.6μL |
| Reverse guideSubstance (0.2. mu. mol/L) | 0.6μL |
| Template DNA (10 ng to 20 ng) | 3μL |
| ddH2O | Adding to 25 μ L |
qPCR amplification was performed on an ABI 7500 PCR apparatus (Thermofeisher, USA) under the conditions of 50 ℃ for 2 min; 95 ℃ for 2min,95 ℃ for 15s,60 ℃ for 1min, 50 ℃ for 1min, for a total of 45 cycles.
c. And (3) judging the PCR result:
positive control: all amplification curves of positive control FAM and VIC signal channels have typical PCR amplification curves, and Ct values are less than or equal to 30;
negative control: negative control FAM and VIC signal channels have no typical PCR amplification curve, and Ct values are both more than or equal to 40 or both negative.
d. And (3) judging polymorphism results:
1) if the detection result has an obvious FAM amplification curve and a typical PCR amplification curve, the Ct value is less than or equal to 35, and the VIC signal channel has no obvious amplification curve, the detection sample is considered to be homozygous insertion mutation.
2) And if the detection result has an obvious VIC amplification curve, the Ct value is less than or equal to 35, and the FAM signal channel has no obvious amplification curve, the detection sample is considered to be the wild type.
3) And if the detection result has an obvious VIC amplification curve, the Ct value is less than or equal to 35, and has an obvious VIC amplification curve, and the Ct value is less than or equal to 35, determining that the detection sample is the DHFR 19bp insertion mutation heterozygous sample.
EXAMPLE III
This example provides a method of analyzing amplification curves for different types of samples.
In the figure, the amplification curve 1 represents that the gene DHFR detects 19bp deletion mutation of the intron, the amplification curve 2 represents that the gene DHFR detects 19bp insertion mutation of the intron, and if the line of the amplification curve parallel to the X axis is considered that the probe signal is not detected (there is no genotype corresponding to the probe).
The method for judging whether the detected sample is a wild type, a homozygous mutant type or a heterozygous type comprises the following steps:
wild type: and if the detection result of the sample is that a deletion signal exists and no insertion signal exists, the two alleles are considered to be deleted, and the type of the sample is judged to be wild type. It should be noted that the DHFR gene functions the worst in the population corresponding to this wild type, with the highest incidence of the associated disease. As shown in FIG. 1, when the amplification curve 2 is parallel to the X-axis, it indicates that no insertion mutation of the 19bp fragment of the intron of the gene DHFR is detected, while when the amplification curve 1 has a large amplification phenomenon, it indicates that the deletion mutation of the 19bp fragment of the intron of the gene DHFR is detected, and the sample detected in FIG. 1 corresponds to the wild-type signal.
Homozygous mutant type: and if the detection result of the sample shows that the insertion signal exists and the deletion signal does not exist, the two alleles are considered to be insertion mutation, and the sample is judged to be homozygous mutation type. It should be noted that the DHFR gene functions best in the population corresponding to this homozygous mutant, with the lowest incidence of the associated disease. As shown in FIG. 2, the amplification curve 1 is parallel to the X-axis, which indicates that no deletion mutation of the 19bp fragment of the intron of the gene DHFR is detected, while the amplification curve 2 shows that the insertion mutation of the 19bp fragment of the intron of the gene DHFR is detected, which corresponds to the wild-type signal detected in the sample of FIG. 2.
Hybrid mutant type: if the sample detection result has both deletion signal and insertion signal, one of the two alleles is considered to be 19bp insertion type, and the other is 19bp deletion type, and the sample is judged to be heterozygous mutant type. It should be noted that the DHFR gene of the population corresponding to the heterozygous mutant has a general function and a moderate incidence of related diseases. As shown in FIG. 3, when both the amplification curve 1 and the amplification curve 2 show a large amplification phenomenon, it indicates that the sample detected in FIG. 3 is a heterozygous signal when the insertion mutation of the 19bp fragment of the intron of the gene DHFR and the deletion mutation of the 19bp fragment of the intron of the gene DHFR are detected.
Sequence listing
<110> Shenzhen Tailede medical Co Ltd
<120> detection method and kit for 19bp insertion mutation of intron of DHFR gene, and use method of kit
<141> 2020-12-29
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ccaaacgggc gcaggc 16
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<212> DNA
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tcgcgcgtcc cgcccaggt 19
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<213> Artificial Sequence (Artificial Sequence)
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agtcggccac cccgaccgt 19
Claims (10)
- A method for detecting 19bp insertion mutation of a DHFR gene intron is characterized in that the method comprises the steps of carrying out real-time fluorescence quantitative PCR on a sample according to a PCR primer combination designed according to the 19bp insertion mutation of the DHFR gene intron and a specific detection fluorescent probe, and further identifying any one of a wild type, a homozygous mutant type and a heterozygous type of the DHFR gene.
- 2. The detection method according to claim 1, wherein the PCR primer combination is:forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3', respectively;reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3' are provided.
- 3. The detection method according to claim 1, wherein the specific detection fluorescent probe is:1, probe 1: a fluorophore 5'-TCGCGCGTCCCGCCCAGGT-3' quencher group;and (3) probe 2: fluorophore 5'-AGTCGGCCACCCCGACCGT-3' quenches the group.
- 4. The detection method according to claim 2, wherein the fluorescent group used at the 5' end of the PCR primer combination is a fluorescent reporter group suitable for fluorescent PCR quantitative analysis, and comprises any one or more of FAM, VIC, TET, HEX and ROX; the 3' quenching group of the PCR primer combination comprises any one or more of BHQ-1, BHQ-2, Dabcy1 and Eclipse.
- 5. A kit is characterized by comprising a PCR primer combination designed by 19bp insertion mutation of a DHFR gene intron and a specific detection fluorescent probe;the PCR primer combination is as follows:forward primer DHFR-F: 5'-CAAAGACCTCACCGGGCA-3';reverse primer DHFR-R: 5'-CCAAACGGGCGCAGGC-3';the specific detection fluorescent probe comprises:1, probe 1: a fluorophore 5'-TCGCGCGTCCCGCCCAGGT-3' quencher group;and (3) probe 2: fluorophore 5'-AGTCGGCCACCCCGACCGT-3' quencher.
- 6. The kit according to claim 5, wherein the fluorescent group used at the 5' end of the PCR primer combination is a fluorescent reporter group suitable for fluorescent PCR quantitative analysis, and comprises any one or more of FAM, VIC, TET, HEX and ROX; the PCR composition comprises 3' quenching groups including any one or more of BHQ-1, BHQ-2, Dabcy1 and Eclipse.
- 7. The kit of claim 5, further comprising a PCR reaction system.
- 8. The kit of claim 7, wherein the PCR reaction system comprises: PCR buffer solution, dNTPs, Taq DNA polymerase and Mg2+Specific amplification primers and deionized water.
- 9. A method of use comprising the kit of any one of claims 5 to 8, comprising the steps of:s1, extracting DNA in the sample;s2, carrying out PCR amplification reaction by using the DNA extracted in the step S1 as a template;and S3, analyzing and judging results.
- 10. The method of using the kit according to claim 9, wherein the conditions of the PCR amplification reaction are as follows: at 50 ℃ for 2min,95 ℃ for 15s,60 ℃ for 1min, 50 ℃ for 1min, for a total of 45 cycles.
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| WO2000052205A2 (en) * | 1999-03-01 | 2000-09-08 | Mcgill University | cDNA FOR HUMAN METHYLENETETRAHYDROFOLATE REDUCTASE |
| CN101173276A (en) * | 2000-12-08 | 2008-05-07 | 茵维特罗根公司 | Compositions and methods for rapidly generating recombinant nucleic acid molecules |
| CN110938682A (en) * | 2019-12-30 | 2020-03-31 | 武汉光谷联合医学检验所股份有限公司 | CYP2C19 gene polymorphism detection kit and application thereof |
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Application publication date: 20220701 |