CN117385010A - qPCR method for detecting gene polymorphism - Google Patents
qPCR method for detecting gene polymorphism Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
Abstract
The invention provides a qPCR method for detecting gene polymorphism, which designs n forward primers, 1 reverse primer and n general sequences with different sequences and a quenching group marked at the 3' end of each allele according to the type n (n is 2, 3 or 4) of the gene polymorphism to carry out amplification reaction; a section of universal nucleotide sequence Q is additionally added to the 5' end of each forward primer, and is respectively reversely complemented with the universal sequence; the 5' end of the general nucleotide sequence Q is marked with a fluorescent group, and the marked fluorescent groups on each general nucleotide sequence are different. The method can accurately detect the polymorphism of the gene by only designing the corresponding number of specific primers with fluorescent group modification and 1 downstream primer according to the polymorphism types and the corresponding number of general sequences with quenching group modification, and does not need to adopt a Taqman probe, and has the advantages of simple operation, rapid detection, clear and accurate interpretation, low cost, universal application and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a qPCR method for detecting gene polymorphism without a fluorescent probe.
Background
Genetic polymorphism is a phenomenon in which two or more alleles exist at a certain locus in the same population, and the frequency of the alleles is greater than 0.01, and the mechanism of formation is gene mutation. The main parameters for evaluating genetic polymorphisms are gene frequency, genotype frequency, and phenotype frequency.
The gene polymorphism may be a single nucleotide polymorphism, a gene insertion, a gene deletion or a difference in the number of short repeated sequences. Single base polymorphisms are most common. The number of biological varieties is large, and the characteristics of each variety are determined by genes. Chemically, a gene consists of a series of base sequences of different arrangements, these bases being A, T or U, C, G. In different individuals of the same species, the gene structure may vary on average by one base per kilobase. The difference between single bases is called a single base polymorphism. The frequency varies from gene to gene, about 0.11% in pseudogenes, about 0.1% in introns and less than 0.1% in exons.
The gene polymorphism analysis and detection has wide theoretical and practical values, including development research, biological evolution research, disease etiology analysis, medicine development, new medicine development, agricultural breeding, etc. The meaning is particularly important in medicine and health. Some genetic diseases are directly related to gene polymorphism and gene expression, and some diseases are related to gene expression and interaction of environmental factors. It is theorized that similar diseases may have similar genetic backgrounds or similar genetic polymorphisms.
Common detection methods for detecting gene polymorphism include Sanger sequencing, electrophoresis, taqman probe fluorescent quantitative PCR, etc. Sanger sequencing is a gold standard for detection, but the method has long period, high cost and low flux, and is difficult to meet the requirements of practical application; the electrophoresis method is simple, low in cost, complex in operation and has the risk of cross contamination; the Taqman probe fluorescent quantitative PCR method is simple to operate and high in flux, but each polymorphic site needs to be synthesized into one MGB probe, so that the cost is high.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a qPCR method for detecting gene polymorphism.
In order to achieve the above purpose, the invention adopts the following technical scheme:
according to a first aspect of the invention, a qPCR method for detecting gene polymorphism is provided, n forward primers, 1 reverse primer and n universal sequences with different sequences and a quenching group marked at the 3' -end of each allele are designed according to the type n (n is one of 2, 3 and 4) of the gene polymorphism to carry out amplification reaction;
a section of universal nucleotide sequence Q is additionally added to the 5' end of each forward primer, and is respectively reversely complemented with the universal sequences; the 5' end of the general nucleotide sequence Q is marked with a fluorescent group, and the marked fluorescent groups on each general nucleotide sequence are different.
Further, the matching degree of the general nucleotide sequence Q and the target sequence to be amplified is lower than 20%, the length is 18-30 bp, and the Tm range is 56-60 ℃.
Further, the fluorescent group is selected from one of FAM, VIC, cy and TET, HEX, JOE, ROX.
Further, the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and DABCYL.
Further, the reaction system used for amplification comprises a primer mixture, 10 Xbuffer, dNTPs, nuclease-free water, taq enzyme and a DNA template.
Further, the procedure used for amplification was as follows: 95 ℃ for 5min; 15s at 95℃and 1min at 60℃for 45 cycles.
In a second aspect, the invention provides the use of the method described above for detecting ACE insertion/deletion polymorphisms using primers and universal sequences of SEQ ID NO: 3-SEQ ID NO: shown in figure 7; the sequence is shown in SEQ ID NO:3 and ID NO:4, respectively labeling different fluorescent groups at the 5' end of the primer; the sequence is shown in SEQ ID NO:6 and ID NO:7 contains a quenching group modification at the 3' end of the general sequence shown in fig.
Further, a reaction system adopted by amplification comprises a primer mixed solution, a 10 Xbuffer solution, dNTPs, nuclease-free water, taq enzyme and a DNA template; the primer mixture is prepared from the following components in concentration: 10. Mu.MACE-I-F primer 1. Mu.l, 10. Mu.MACE-D-F primer 0.2. Mu.l, 10. Mu.MACE-D/I-R primer 1.2. Mu.l, 10. Mu.M universal sequence N10.2. Mu.l and 10. Mu.M universal sequence N21. Mu.l.
Further, the reaction system used for amplification included 3.6. Mu.l of primer mix, 2.0. Mu.l of 10 Xbuffer, 1.6. Mu.l of 2.5mM dNTP, 11.6. Mu.l of nuclease-free water, 0.2. Mu.l of Taq enzyme and 1.0. Mu.l of 10 ng/. Mu.l of DNA template.
In a third aspect, the invention provides a kit for detecting an ACE insertion/deletion polymorphism, comprising a nucleic acid sequence as set forth in SEQ ID NO: 3-SEQ ID NO:7 and universal sequences; the sequence is shown in SEQ ID NO:3 and ID NO:4, respectively labeling different fluorescent groups at the 5' end of the primer; the sequence is shown in SEQ ID NO:6 and ID NO:7 contains a quenching group modification at the 3' end of the general sequence shown in fig.
Further, the fluorescent group is selected from one of FAM, VIC, cy and TET, HEX, JOE, ROX.
Further, the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA and DABCYL.
Compared with the prior art, the invention has the following technical effects:
the qPCR method for detecting the gene polymorphism provided by the invention can accurately detect the gene polymorphism by only designing the corresponding number of specific primers with fluorescent group modification and 1 downstream primer and the corresponding number of general sequences with quenching group modification according to the polymorphism types, and does not need to adopt a Taqman probe, and has the advantages of simplicity in operation, rapidness in detection, clear and accurate interpretation, low cost, universal application and the like.
Drawings
FIG. 1 is a schematic diagram of the primer design principle of the qPCR method provided by the invention;
FIG. 2 is a graph showing the results of detecting peripheral blood genomic DNA (ACE-type I) according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of detecting peripheral blood genomic DNA (ACE-D type) according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of detecting peripheral blood genomic DNA (ACE-I/D heterozygous) in an embodiment of the present invention;
FIG. 5 is a graph showing the results of detecting genomic DNA from 10-exception peripheral blood by electrophoresis (type I: 490bp band; type D: 190bp band; type D/I: 190bp/490bp double band).
Detailed Description
The invention provides a qPCR method for detecting gene polymorphism without a Taqman probe, which comprises the steps of designing n forward primers, 1 reverse primer and n general sequences with different sequences and a quenching group marked at the 3' -end of each allele according to the type n (n is one of 2, 3 and 4) of the gene polymorphism to carry out amplification reaction;
a section of universal nucleotide sequence Q is additionally added to the 5' end of each forward primer, and is respectively reversely complemented with the universal sequences; the 5' end of the general nucleotide sequence Q is marked with a fluorescent group, and the marked fluorescent groups on each general nucleotide sequence are different. For example, for a gene (see FIG. 1), the allele number is 2, and the primer design principle is as follows:
2 forward primers capable of specifically recognizing allele 1 and allele 2, 1 reverse primer, and 2 universal sequences N1 and N2 were designed.
The allele 1 forward primer is characterized by: the nucleotide sequence can specifically amplify the allele 1, and a section of general nucleotide sequence Q1 is additionally added to the 5' end of the nucleotide sequence. The Q1 sequence is characterized by: the matching degree of the nucleotide sequence and the amplified target sequence is lower than 20%, the length is 18-30 bp, the Tm range is 56-60 ℃, and the 5' end is marked with FAM or other fluorescent group modification; the allele 2 forward primer is characterized by: the nucleotide sequence can specifically amplify the allele 2, and a section of general nucleotide sequence Q2 is additionally added to the 5' end of the nucleotide sequence. The Q2 sequence is characterized by: the matching degree of the nucleotide sequence and the amplified target sequence is lower than 20%, the length is 18-30 bp, the Tm range is 56-60 ℃, HEX or other fluorescent group modification (different from Q1 fluorescent group) is marked at the 5' end of Q2. The N1 universal sequence is characterized by: the nucleotide sequence is reversely complementary to Q1, and the 3' end contains BHQ1 or other quenching group modification; the N2 universal sequence is characterized by: the nucleotide sequence is complementary to Q2 in reverse and contains a modification of BHQ1 or other quenching groups at its 3' end.
Based on the principle design, before qPCR amplification, the sequence of the N1 general sequence and the sequence of the Q1 of the allele 1 forward primer are reversely complementary, the sequence of the N2 general sequence and the sequence of the Q2 of the allele 2 forward primer are reversely complementary, the fluorescent signal emitted by the Q1 fluorescent gene is quenched by the quenching group of N1, and the fluorescent signal emitted by the Q2 fluorescent gene is quenched by the quenching group of N2, so that no fluorescent signal is generated. In qPCR amplification, a high temperature denaturation step results in N1 being separated from Q1, N2 being separated from Q2, and allele forward primer 1 and allele forward primer 2 being capable of binding to the corresponding DNA templates, together with the reverse primer, for PCR amplification, thereby generating a fluorescent signal. The result can be interpreted according to the qPCR amplification curve: only FAM fluorescence curves, called allele 1; only the HEX fluorescence curve, designated allele 2; if there is both FAM and HEX fluorescence curves, it is referred to as allele type 1/2.
In a preferred embodiment of the invention, the universal nucleotide sequence Q has a degree of match with the target sequence to be amplified of less than 20%, a length of 18 to 30bp and a Tm of 56 to 60 ℃.
In a preferred embodiment of the present invention, the fluorescent group is selected from one of FAM, VIC, cy, TET, HEX, JOE, ROX.
In a preferred embodiment of the present invention, the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL.
In a preferred embodiment of the invention, the reaction system used for amplification comprises a primer mix, 10 Xbuffer, dNTPs, nuclease-free water, taq enzyme and DNA template.
In a preferred embodiment of the invention, the amplification is performed using the following procedure: 95 ℃ for 5min; 15s at 95℃and 1min at 60℃for 45 cycles.
In a second aspect, the invention provides the use of the method described above for detecting ACE insertion/deletion polymorphisms using primers and universal sequences of SEQ ID NO: 3-SEQ ID NO: shown at 7.
In a preferred embodiment of the invention, the reaction system used for amplification comprises primer mixture, 10 Xbuffer, dNTP, nuclease-free water, taq enzyme and DNA template; the primer mixture is prepared from the following components in concentration: 10. Mu.MACE-I-F primer 1. Mu.l, 10. Mu.MACE-D-F primer 0.2. Mu.l, 10. Mu.M ACE-D/I-R primer 1.2. Mu.l, 10. Mu.M universal sequence N10.2. Mu.l and 10. Mu.M universal sequence N21. Mu.l.
In a preferred embodiment of the invention, the reaction system used for amplification comprises 3.6. Mu.l of primer mix, 2.0. Mu.l of 10 Xbuffer, 1.6. Mu.l of 2.5mM dNTPs, 11.6. Mu.l of nuclease-free water, 0.2. Mu.l of Taq enzyme and 1.0. Mu.l of 10 ng/. Mu.l of DNA template.
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
The effect of the ACE insertion/deletion polymorphism verification method is detected in the embodiment, the ACE (Angiotensin-converting enzyme) gene is located on human chromosome 17, and the specifically encoded ACE protein consists of 259 amino acids, can catalyze the conversion of angiotensinogen (Angiotensin I) into Angiotensin II (Angiotensin II), and is an important component of human immunity and vascular response mechanism. The research on the relation between ACE gene insertion/deletion (I/D) polymorphism and various diseases relates to a plurality of fields, including various processes, symptoms, prognosis, response to drugs and the like of diseases, and most researches consider that the ACE gene polymorphism is related to coronary heart disease, myocardial infarction, left ventricular hypertrophy, type II diabetes, atherosclerosis, diabetic nephropathy and other diseases. The specific detection steps and results are as follows:
1. sample DNA extraction
The DNA extraction was performed on the peripheral blood of 10 normal persons using the "blood genome column type miniprep kit" from century corporation, and then the DNA concentration and quality detection was performed using Nanodrop, and finally diluted to a concentration of 10 ng/. Mu.l.
Preparation of PCR System
2.1 preparation of ACE primer mixture
Firstly, 2 specific primers with fluorescent group modification, 1 downstream primer and 2 general sequences with quenching group modification are dissolved into 10 mu M concentration by deionized water, and ACE primer mixed solution is prepared according to the proportion of the table 1.
D-type reference sequence (corresponding or complementary to the upstream and downstream primers at the horizontal line)
CTGCTGCCTATACAGTCATTTTATGTGGTTTCGCCAATTTTATTCCAGCCTCTGAAATTCTCTGAGCTCCCCTTACAAGCAGAGGTGAGCTAAGGGCTGGAG(SEQ ID NO：1)
Type I reference sequence (corresponding or complementary to the upstream and downstream primers at the horizontal line)
CAGGCGTGATACAGTCACTTTTATGTGGTTTCGCCAATTTTATTCCAGCTCTGAAATTCTCTGAGCTCCCCTTACAAGCAGAGGTGAGCTAAGGGCTGGAGC(SEQ ID NO：2)
ACE-D-F primer (modified 5' HEX, general nucleotide sequence Q1 at horizontal line)
TGATACGGCGACCACCGCTGCTGCCTATACAGTCACTTTTATG(SEQ ID NO：3)
ACE-I-F primer (5' FAM modification, common nucleotide sequence Q2 at the horizontal line)
CAAGCAGAAGACGGCATACGCAGGCGTGATACAGTCACTTTTATG(SEQ ID NO：4)
ACE-D/I-R primer:
TTAGCTCACCTCTGCTTGTAAGG(SEQ ID NO：5)
n1 general sequence (3' BHQ1 modification)
CGGTGGTCGCCGTATCA(SEQ ID NO：6)
N2 general sequence (3' BHQ1 modification)
CGTATGCCGTCTTCTGCTTG(SEQ ID NO：7)
TABLE 1 proportioning of ACE primer mixture
| Primer name | Primer concentration | Volume (mul) |
| ACE-I-F | 10μM | 1 |
| ACE-D-F | 10μM | 0.2 |
| ACE-D/I-R | 10μM | 1.2 |
| N1 | 10μM | 0.2 |
| N2 | 10μM | 1 |
2.2 preparation of amplification System: the amplification system shown in Table 2 was used for the preparation.
TABLE 2 ACE amplification System (20 μl)
| The components | Volume (mul) |
| 10 Xbuffer | 2.0 |
| 2.5mM dNTP | 1.6 |
| ACE primer mixture | 3.6 |
| Nuclease-free water | 11.6 |
| Taq enzyme | 0.2 |
| DNA template (10 ng/. Mu.l) | 1.0 |
Qpcr amplification: qPCR amplification was performed according to the amplification procedure shown in Table 3.
TABLE 3 PCR amplification procedure
4. Interpretation and description of results
And judging the result according to a qPCR amplification curve: only FAM fluorescence curve, designated type I (shown in fig. 2); only the HEX fluorescence curve, designated as type D (shown in fig. 3); if there is both FAM and HEX fluorescence curves, this is called type I/D (shown in FIG. 4). 10 samples were simultaneously tested by agarose gel electrophoresis (FIG. 5): only 490bp electrophoresis band, designated type I; only 190bp of electrophoresis band, called D-type; if there is both a 190bp and 490bp band, it is called D/I.
The results of comparison of the invention with electrophoresis are shown in Table 4 below:
TABLE 4 comparison of the detection results of the invention with the electrophoresis method
| Sample numbering | Results of the invention | Electrophoresis results |
| 1 | I | I |
| 2 | D/I | D/I |
| 3 | D | D |
| 4 | I | I |
| 5 | D/I | D/I |
| 6 | I | I |
| 7 | I | I |
| 8 | D/I | D/I |
| 9 | I | I |
| 10 | I | I |
As can be seen from Table 4, the consistency of the detection result of the method provided by the invention and the electrophoresis method is 100%, and the detection result is accurate and reliable. Compared with the electrophoresis method, the method has no risk of cross contamination and is convenient to operate; the method provided by the invention does not need to synthesize the Taqman probe, and can be used for detecting other alleles after the N1 and N2 general sequences are synthesized, so that the cost is lower than that of the traditional Taqman probe qPCR method.
In conclusion, the qPCR method for detecting the gene polymorphism has the advantages of simplicity in operation, rapidness in detection, clearness and accuracy in interpretation, low cost, universal application and the like.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (10)
1. A qPCR method for detecting gene polymorphism is characterized in that n forward primers, 1 reverse primer and n universal sequences with different sequences and a quenching group marked at the 3' -end are designed according to the type n (n is one of 2, 3 and 4) of the gene polymorphism to carry out amplification reaction;
a section of universal nucleotide sequence Q is additionally added to the 5' end of each forward primer, and is respectively reversely complemented with the universal sequence; the 5' end of the general nucleotide sequence Q is marked with a fluorescent group, and the marked fluorescent groups on each general nucleotide sequence are different.
2. The qPCR method according to claim 1, wherein the universal nucleotide sequence Q matches less than 20% with the target sequence to be amplified, is 18-30 bp in length, and has a tm in the range of 56-60 ℃.
3. The qPCR method according to claim 1, wherein the fluorophore is selected from one of FAM, VIC, cy, TET, HEX, JOE, ROX.
4. The qPCR method according to claim 1, wherein the quenching group is selected from one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL.
5. The qPCR method according to claim 1, wherein the reaction system used for the amplification comprises a primer mix, 10 x buffer, dntps, nuclease-free water, taq enzyme and DNA template.
6. The qPCR method according to claim 1, wherein the amplification is performed using the following procedure: 95 ℃ for 5min; 15s at 95℃and 1min at 60℃for 45 cycles.
7. Use of the method according to any one of claims 1-6 for detecting ACE insertion/deletion polymorphisms, wherein the primers and universal sequences used are as set forth in SEQ ID NO: 3-SEQ ID NO: shown in figure 7; the sequence is shown in SEQ ID NO:3 and ID NO:4, respectively labeling different fluorescent groups at the 5' end of the primer; the sequence is shown in SEQ ID NO:6 and ID NO:7 contains a quenching group modification at the 3' end of the general sequence shown in fig.
8. The use according to claim 7, wherein the reaction system for amplification comprises a primer mix, 10 Xbuffer, dNTPs, nuclease-free water, taq enzyme and DNA template; the primer mixture is prepared from the following components in concentration: 10. Mu.MACE-I-F primer 1. Mu.l, 10. Mu.MACE-D-F primer 0.2. Mu.l, 10. Mu.MACE-D/I-R primer 1.2. Mu.l, 10. Mu.M universal sequence N10.2. Mu.l and 10. Mu.M universal sequence N21. Mu.l.
9. The method of claim 7, wherein the reaction system comprises 3.6. Mu.l of primer mixture, 2.0. Mu.l of 10 Xbuffer, 1.6. Mu.l of 2.5mM dNTP, 11.6. Mu.l of nuclease-free water, 0.2. Mu.l of Taq enzyme and 1.0. Mu.l of 10 ng/. Mu.l of DNA template.
10. A kit for detecting ACE insertion/deletion polymorphisms, comprising a sequence as set forth in SEQ ID NO: 3-SEQ ID NO:7 and universal sequences; the sequence is shown in SEQ ID NO:3 and ID NO:4, respectively labeling different fluorescent groups at the 5' end of the primer; the sequence is shown in SEQ ID NO:6 and ID NO:7 contains a quenching group modification at the 3' end of the general sequence shown in fig.
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