CN111073975A - Probe system for gene SNP locus detection and application thereof - Google Patents
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Abstract
The invention discloses a probe system for gene SNP locus detection and application thereof, wherein the probe system at least comprises 2 probe pairs; each probe pair comprises an oligonucleotide sequence modified by a 5 'end fluorescent group and an oligonucleotide sequence modified by a corresponding complementary combined 3' end quenching group; the fluorophore for each probe pair is different; the Tm value of the oligonucleotide sequence modified by the 5 'end fluorescent group does not exceed the Tm value of the oligonucleotide sequence modified by the 3' end quenching group. The invention also provides a reaction system for detecting the gene SNP locus, a detection method of the gene SNP locus and application of the probe system in detection of the deaf gene SNP locus, wherein the method is used for detecting the deaf gene related SNP locus, and the detection accuracy of 16 deaf gene related SNP loci is up to 100%.
Description
Technical Field
The invention relates to the fields of biotechnology and medicine, in particular to a probe system for detecting gene SNP sites and application thereof.
Background
A Single Nucleotide Polymorphism (SNP) site refers to a difference between one base and one base between humans, and is a DNA sequence polymorphism caused by variation at the genomic nucleotide level, and the genomic sequence difference includes single base transition, transversion, single base insertion and deletion, and the like. More than 99.9% of the human gene sequences are identical and only 0.1% of the sequences are different, namely 1 of 1000 bases is different, and the human genome has 30 hundred million pairs of bases, so that the total number of the bases in the human genome is 300 ten thousand bases. Generally, the 300 ten thousand different bases are called SNP, which is determined by the individual genetic background, in the current research, SNP locus can be used as a clinically meaningful diagnostic Marker (Marker), which has great clinical significance, and can be used for discovery of high-risk groups, identification of disease-related genes, drug design and test, basic research of biology, and the like.
The current methods for detecting SNP sites mainly comprise a sequencing method, a chip method and a Taqman method. Wherein, the sequencing method is very accurate, but the price is also very high, and the method is not suitable for the detection of large sample size; the chip method is suitable for ultra-multi-site analysis, but the chip design cost is high, and some SNPs cannot be detected due to the complexity of DNA samples, so the accuracy is low; the Taqman method is high in accuracy, suitable for large samples, and expensive. Therefore, it is very important to develop a method for detecting gene SNP sites, which is low in cost, high in efficiency and suitable for large sample detection.
According to the latest statistical data of the national disabled people survey in 2006, the number of hearing speech disabled people in China reaches 2780 thousands, about 3 thousands of deaf children are born each year, and 6-8 thousands of sick children suffering from delayed deafness are found, wherein most of the deafness is severe or extremely severe sensorineural deafness. This type of deafness severely affects the communication and cognitive abilities of the patient, placing a tremendous burden on the individual, family, and society.
With the completion of the human genome project, the etiology of genetic deafness has been studied in great progress, and it is presumed that the congenital sensorineural deafness of 2/3 is caused by genetic factors. Non-syndromic deafness accounts for 70% of all hereditary deafness, 80% of which are autosomal recessive inheritance, 15% are autosomal dominant inheritance, and the other 5% are mitochondrial or X-linked inheritance. Although many genes are involved in deafness, most deaf patients are caused by the defect of a few single genes. The molecular diagnosis center for deafness of the general hospital of the liberation military carries out the national molecular epidemiological investigation of deafness from 2004, determines that GJB2, SLC26A4 and mitochondrial gene mtDNA (A1555G and C1494T mutations) are the 3 most common deafness genes causing most hereditary deafness in China, and finds that 21% of deafness patients have GJB2 mutation; 14.5% of patients harbored the SLC26A4 mutation, and 3.4% and 0.6% of patients harbored the mtDNA A1555G and C1494T mutations, respectively. The GJB2 mutation mainly causes congenital non-syndromic deafness, and is an autosomal recessive genetic pattern. The major mutation was 235 delC. Through the GJB2 deafness which is clearly diagnosed through the gene, on the basis of clear causes of diseases, other nervous system diseases can be eliminated, the completeness of cochlear nerve and auditory center is prompted, and multiple studies show that the hearing language rehabilitation effect of children suffering from the diseases after the children are implanted into the artificial cochlea is good, so that the method is an ideal effect prediction examination before the artificial cochlea operation. The SLC26a4 mutation may cause two clinical manifestations, one is Pendred syndrome, manifested as goiter and deafness; the other is most common in China, the infant only shows deafness, and the CT examination can find that the vestibular aqueduct is enlarged, which is called large vestibular aqueduct syndrome (EVAS). The EVAS accounts for 20-28% of the incidence rate of the children deafness in China. The reason for the disease is that because the channel connecting the cranial cavity and the inner ear is abnormally enlarged, the EVAS children can have hearing loss caused by factors causing intracranial pressure change, such as mild head collision, cold and the like. In EVAS deafness patients in China, the detection rate of SLC26A4 gene mutation reaches about 97%, and the main mutation is c.919A > G. Deafness caused by mutation of mitochondrial gene mtDNA A1555G and C1494T is mainly related to aminoglycoside drug use. Mutation carriers are abnormally sensitive to aminoglycoside drugs, and tinnitus and even severe hearing loss can occur with low dose. Since it follows the manner of maternally inheritance, i.e., the inheritance of such mutant genes is only directly transmitted to offspring by women, if there are 1 in a family, it can be deduced that there are at least 10 in this family carry such mutant genes. If mother carries the mutant gene, the mother can remind himself and the offspring that aminoglycoside medicine should be forbidden to use, so as to avoid deafness; meanwhile, 1 case diagnosed in the family can be used as the medication guide of the whole family, thereby blocking the deafness from continuing to occur in the family.
Therefore, by screening the deafness gene SNP, screened mtDNA A1555G and C1494T mutation carriers can effectively avoid drug-induced deafness by prohibiting the use of aminoglycoside antibiotics; the GJB2 deaf patient indicates that the artificial cochlea has good prognosis and can directly enter the hearing recovery procedure. SLC26A4 deaf patient, indicate to be EVAS infant, through taking strict safeguard procedures, can keep the infant as far as possible to have effective hearing. And the deafness gene diagnosis can also provide scientific and accurate genetic information and guidance for the marriage and breeding of deaf patients and deaf families.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a probe system for detecting the gene SNP locus, which can efficiently detect the gene SNP locus for genotyping.
The invention also provides a reaction system for detecting the gene SNP locus.
The invention also provides a detection method of the gene SNP locus.
The invention also provides the application of the detection method of the gene SNP locus.
According to the first aspect of the invention, the probe system for gene SNP site detection comprises at least 2 probe pairs; each probe pair comprises an oligonucleotide sequence modified by a 5 'end fluorescent group and an oligonucleotide sequence modified by a corresponding complementary combined 3' end quenching group; the fluorophore for each probe pair is different; the Tm value of the oligonucleotide sequence modified by the 5 'end fluorescent group does not exceed the Tm value of the oligonucleotide sequence modified by the 3' end quenching group.
The probe system according to the embodiment of the first aspect of the invention has at least the following beneficial effects: the same probe system can be used for different SNP sites, and compared with a Taqman method, the detection cost, particularly the research and development cost is greatly saved.
According to some embodiments of the invention, the probe pair is selected from the group consisting of 4 probe pairs, probe pair 1 comprising an oligonucleotide sequence modified with a fluorophore at the 5 'end of the nucleotide sequence SEQ ID No.1 and an oligonucleotide sequence modified with a quencher at the 3' end of the nucleotide sequence SEQ ID No. 2;
the probe pair 2 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.3 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 4;
the probe pair 3 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.5 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 6;
the probe pair 4 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.7 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 8.
According to some embodiments of the invention, the fluorophore modification comprises at least one of FAM, TET, JOE, HEX, CY3, CY5, ROX, and TAMRA; the quencher modification includes at least one of BHQ-1 and BHQ-2.
A reaction system for gene SNP site detection according to an embodiment of the second aspect of the invention, the reaction system comprises the probe system and a primer set designed for the SNP site; each primer group comprises 1 wild type primer sequence and 1 mutant type primer sequence; the wild type primer and the mutant type primer both contain oligonucleotide sequences corresponding to probes with fluorescent groups in the probe pairs.
Preferably, the oligonucleotide sequences of the primer set consist of oligonucleotide sequences corresponding to probes modified with 5' terminal fluorophores and SNP site-specific primer sequences.
The reaction system according to the embodiment of the second aspect of the present invention has at least the following beneficial effects: the probe system contained in the reaction system can be suitable for detecting different SNP sites, has the advantages of low price and high efficiency, and has high detection accuracy and convenient use when being used for detecting the SNP sites; the invention adopts high-fidelity DNA polymerase with 3 ' to 5 ' exonuclease activity and a primer modified by 3 ' end sulfurated phosphoric acid, which can greatly reduce non-specific amplification, thereby accurately typing SNP loci.
According to some embodiments of the invention, the 3' terminal base or adjacent bases of the wild type primer sequence and the mutant primer sequence are modified with phosphorothioate; the sulfuration modified base pair exonuclease digestion has certain tolerance.
According to some embodiments of the invention, the reaction system further comprises a high fidelity DNA polymerase; primers which are not modified by the sulfurized phosphate cannot tolerate the digestion of exonuclease, and when mismatch occurs, high-fidelity DNA polymerase can cut off mismatched bases and continue extension; and the primer modified by 3' end sulfurated phosphate can resist exonuclease digestion, and when mismatching occurs, mismatching bases cannot be cut off in time, so that polymerization reaction is terminated, and extension cannot be carried out. Thus, efficient extension can only be performed when the primer and template are perfectly matched, thereby reducing non-specific amplification.
Preferably, the high fidelity polymerase includes at least one of Pfu polymerase and Deep Vent polymerase.
Preferably, the amounts of the components of the reaction system are as follows: the concentration of each primer in the primer group is 0.05-0.25 mu M; in the probe system, the concentration of the oligonucleotide sequence modified by the 5 'end fluorescent group is 0.1 mu M, and the concentration of the oligonucleotide sequence modified by the 3' end quenching group is 0.15 mu M.
According to the third aspect of the invention, the method for detecting the SNP locus of the gene comprises the following steps:
s1, constructing the reaction system aiming at each SNP locus;
and S2, carrying out PCR reaction to obtain a detection result.
The detection method according to the embodiment of the third aspect of the invention has at least the following beneficial effects: the invention can adopt 4 channels to detect 2 SNP sites simultaneously, and compared with the conventional 2 channels to detect 1 SNP site, the flux is doubled.
According to some embodiments of the invention, the procedure of the PCR reaction is: 15min at 94 ℃; 94 ℃ for 20s, 60 ℃ for 60s, 10 cycles; collecting fluorescence at 94 ℃ for 20s and 55 ℃ for 60s, and collecting 35 cycles; fluorescence was collected at 30 ℃ for 60 s.
According to the application of the fourth aspect of the embodiment of the invention, the method is applied to detecting the SNP sites related to the deafness gene.
The application of the embodiment of the fourth aspect of the invention has at least the following beneficial effects: the 16 SNP loci of the deafness related susceptibility gene selected by the invention basically cover the main deafness susceptibility gene SNP loci in Chinese population, the detection method provided by the invention can be used for accurately typing the SNP related to the deafness gene, and the detection method has the advantages of low price, high efficiency in use and high accuracy.
According to some embodiments of the invention, the deafness gene-related SNP sites include 16 SNP sites associated with four deafness genes located at GJB2, GJB3, SLC26a4 and MT-RNR 1; the 16 SNP sites comprise c.176-191del16b, c.35delG, c.299-300delAT, c.235delC and 512insAACG on the GJB2 gene, c.538C > T on the GJB3 gene, c.919-2A > G, c.2027T > A, c.2168A > G, c.1174A > T, c.1226G > A, c.1229C > T, 1707+5G > A, c.1975G > C and m.1555A > G and m.1495394C > T on the MT-RNR 2 (12S rRNA) gene.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the detection principle in example 1 of the present invention, wherein F represents a fluorophore FAM, H represents a fluorophore HEX, and B represents a quencher BHQ 2;
FIG. 2 shows the result of typing GJB2-c.176-191del16bp in example 2 of the present invention;
FIG. 3 shows the result of typing GJB2-c.35delG in example 2 of the present invention;
FIG. 4 shows the results of the GJB3-c.538C > T typing in example 2 of the present invention;
FIG. 5 shows the results of typing SLC26A4-c.919-2A > G in example 2 of the present invention;
FIG. 6 shows the results of typing SLC26A4-c.2027T > A in example 2 of the present invention;
FIG. 7 shows the results of SLC26A4-c.2168A > G typing in example 2 of the present invention;
FIG. 8 shows the typing results of GJB2-c.235delC in example 3 of the present invention;
FIG. 9 shows the results of typing SLC26A4-c.919-2A > G in example 3 of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
Example 1: design of universal probe and primer for detecting deafness gene locus
1. Design idea
Designing a probe system: in total, 4 probe pairs were designed, each probe pair comprising an oligonucleotide sequence modified with a 5 'terminal fluorophore and an oligonucleotide sequence modified with a corresponding complementary binding 3' terminal quencher group, the fluorophores of each probe pair being different.
The design method of the probe and the primer comprises the following steps:
(1) each probe pair comprises an oligonucleotide sequence modified by a 5 'end fluorescent group and an oligonucleotide sequence modified by a 3' end quenching group, and the two sequences can be partially or completely combined in a complementary way to form a luminescence-quenching probe system.
(2) The 5 ' terminal fluorophore modified oligonucleotide sequence and the 3 ' terminal quencher modified oligonucleotide sequence of each probe pair may have different Tm values, and the Tm value of the 5 ' terminal fluorophore modified oligonucleotide sequence may be the same as the Tm value of the 3 ' terminal quencher modified oligonucleotide sequence or lower than the Tm value of the 3 ' terminal quencher modified oligonucleotide sequence.
(3) The oligonucleotide sequence of the primer group consists of an oligonucleotide sequence modified by a 5' end fluorescent group and an SNP site-specific primer sequence. The Tm value of the SNP site-specific primer sequence is higher than that of the oligonucleotide sequence modified with a 5' -terminal fluorescent group.
Designing a related SNP locus detection primer group: the oligonucleotide sequence of the primer group consists of an oligonucleotide sequence modified by a 5' end fluorescent group and an SNP site-specific primer sequence. The primer set comprises a wild primer sequence and a mutant primer sequence. When the wild-type primer or the mutant primer is completely matched with the template, a polymerization reaction is carried out to generate a product matched with the oligonucleotide sequence modified by the 5 'end fluorescent group, and when the product is combined with the oligonucleotide sequence modified by the 5' end fluorescent group, a corresponding detectable fluorescent signal is emitted. 3' terminal bases or adjacent bases of the wild type primer sequence and the mutant type primer sequence are modified by sulfurated phosphoric acid, and the base pair exonuclease digestion after sulfuration modification has certain tolerance.
The primer is modified by sulfurated phosphate during oligonucleotide synthesis, and the effects of resisting exonuclease digestion and prolonging the half-life period of the oligonucleotide can be achieved. Zhang Jia et al found that when mismatch occurs in high fidelity DNA polymerase, the polymerase cannot be repaired or repaired in time, the non-mature termination of the polymerization reaction and the extension cannot be performed by comparing the extension reaction of the high fidelity DNA polymerase containing 3 ' to 5 ' exonuclease activity with the low fidelity DNA polymerase not containing 3 ' to 5 ' exonuclease activity on the 3 ' incomplete pairing primer. By utilizing the characteristic of high fidelity enzyme, wild type primers and mutant type primers modified by end phosphorothioate are designed, and single nucleotide polymorphism of corresponding sites can be detected by a luminescence-quenching probe system.
The detection principle of the scheme is shown in figure 1, in the system, when a wild type primer or a mutant type primer is completely matched with a template, a polymerization reaction is carried out to generate a product matched with the oligonucleotide sequence modified by the 5 'end fluorescent group, and when the product is combined with the oligonucleotide sequence modified by the 5' end fluorescent group, a corresponding detectable fluorescent signal is emitted.
2. Designed probes and primers
The probe and primer sequences were designed as shown in table 1 below:
TABLE 1 primer and Probe sequence Listing
Example 2: double system test
The fluorescent quantitative PCR reaction is respectively carried out on wild type leucocytes, plasmids and corresponding mixed samples of the leucocytes and the plasmids of 6 sites of deafness genes GJB2-c.176-191del16bp, GJB2-c.35delG, GJB3-c.538C > T, SLC26A4-c.919-2A > G, SLC26A4-c.2027T > A, SLC26A4-c.2168A > G by using a CY5/ROX double probe.
1. Testing a sample:
a: human leukocyte DNA;
b: a GJB2 mutant plasmid;
c: a GJB3 mutant plasmid;
d: SLC26a4 mutant plasmid 1;
e: SLC26a4 mutant plasmid 2;
g: human leukocyte DNA mixed with the GJB2 mutant plasmid;
h: human leukocyte DNA mixed with the GJB3 mutant plasmid;
i: human leukocyte DNA mixed with SLC26a4 mutant plasmid 1;
j: human leukocyte DNA mixed with SLC26a4 mutant plasmid 2;
k: human leukocyte DNA mixed with SLC26a4 mutant plasmid 3;
the samples were diluted to 5 ng/. mu.L after concentration determination.
2. Reaction system:
preparing a reaction system (the unit is mu L) by using a premixed enzyme buffer required by the reaction, a primer for specific site amplification and a common probe according to the following table 2:
TABLE 2. Dual reaction System
| |
2 |
| 2× |
5 |
| WF | 0.1 |
| MF | 0.1 |
| R | 0.25 |
| CY5-P1 | 0.1 |
| P2-BHQ2 | 0.15 |
| ROX-P3 | 0.1 |
| P4-BHQ2 | 0.15 |
| ddH2O | 2.05 |
| Total | 10 |
In Table 2, WF is the wild type primer, MF is the mutant type primer, and R is the common primer, corresponding to the primer and probe sequences in example 1, respectively.
The sample was placed in a fluorescence quantitative reaction apparatus (Bosun LineGene9600) and reacted according to the following procedure.
3. Reaction procedure:
15min at 94 ℃; {94 ℃ 20s, 60 ℃ 60s, 10cycles }; {94 ℃ 20s, 55 ℃ 60s, fluorescence collection, 30cycles }; fluorescence was collected at 30 ℃ for 60 s.
4. And (4) analyzing results:
the typing results of GJB2-c.176-191del16bp are shown in FIG. 2;
the typing results of GJB2-c.35delG are shown in FIG. 3;
the typing results of GJB3-c.538C > T are shown in FIG. 4;
SLC26A4-c.919-2A > G typing results are shown in FIG. 5;
the results of SLC26A4-c.2027T > A typing are shown in FIG. 6;
SLC26A4-c.2168A > G typing results are shown in FIG. 7;
in FIGS. 2 to 7, allele 1 represents the wild type, allele 2 represents the mutant type, and heterozygosity represents the heterozygous type.
The graph analysis shows that the deafness gene GJB2-c.176-191del16bp, GJB2-c.35delG, GJB3-c.538C > T, SLC26A4-c.919-2A > G, SLC26A4-c.2027T > A, SLC26A4-c.2168A > G6 sites of the deafness gene carry out fluorescent quantitative reaction on wild type leucocytes, plasmids and corresponding mixed samples of leucocytes and plasmids, and the typing is correct.
Example 3: quadruple system test
And performing fluorescent quantitative PCR reaction on a sample of which the deafness genes GJB2-c.235delC and SLC26A4-c.919-2A > G are sequenced to determine the SNP type by using a CY5/ROX/FAM/HEX quadruple probe.
1. Testing a sample:
a is leukocyte DNA;
c.235delC heterozygous sample;
c.235delC pure mutation sample;
d, c.919-2A > G heterozygous sample;
samples A to D above were diluted to 5ng/uL after concentration determination.
2. Reaction system:
preparing a reaction system (the unit is mu L) by using a premixed enzyme buffer required by the reaction, a primer for specific site amplification and a common probe according to the following table 3:
TABLE 3 quadruple reaction system
| |
2 |
| 2× |
5 |
| WF1 | 0.1 |
| MF1 | 0.1 |
| R1 | 0.25 |
| WF2 | 0.1 |
| MF2 | 0.1 |
| R2 | 0.25 |
| CY5-P1 | 0.1 |
| P2-BHQ2 | 0.15 |
| ROX-P3 | 0.1 |
| P4-BHQ2 | 0.15 |
| FAM-P5 | 0.1 |
| P6-BHQ2 | 0.15 |
| HEX-P7 | 0.1 |
| P8-BHQ2 | 0.15 |
| ddH2O | 1.1 |
| |
10 |
In Table 3, WF is the wild type primer, MF is the mutant type primer, and R is the common primer, corresponding to the primer and probe sequences in example 1, respectively.
The sample was placed in a fluorescence quantitative reaction apparatus (Bosun LineGene9600) and reacted according to the following procedure.
3. Reaction procedure:
15min at 94 ℃; {94 ℃ 20s, 60 ℃ 60s, 10cycles }; {94 ℃ 20s, 55 ℃ 60s, fluorescence collection, 35cycles }; fluorescence was collected at 30 ℃ for 60 s.
4. And (4) analyzing results:
the typing results of GJB2-c.235delC are shown in FIG. 8;
SLC26A4-c.919-2A > G typing results are shown in FIG. 9;
in FIGS. 8 to 9, allele 1 represents the wild type, allele 2 represents the mutant type, and heterozygosity represents the heterozygous type.
As can be seen from the graph analysis, the quadruple system of the deafness genes GJB2-c.235delC and SLC26A4-c.919-2A & gtG has correct detection results for the typing of wild type leucocyte, c.235delC heterozygous sample, c.235delC pure mutant sample and c.919-2A & gtG heterozygous sample.
Example 4: clinical sample detection of deafness
12 cases of deafness gene pure mutation or mutation carriers with SNP types determined by sequencing are detected, and the specific conditions are shown in the following table 4:
TABLE 4 genotype of samples
| Sample numbering | Gene | Mutation | Sequencing genotype | |
| 1 | GJB2 | | Hybrid | |
| 2 | GJB2 | | Hybrid | |
| 3 | GJB2 | | Hybrid | |
| 4 | GJB2 | c. | Homozygous mutations | |
| 5 | GJB2 | c. | Homozygous mutations | |
| 6 | GJB2 | c.35delG | Hybrid | |
| 7 | GJB2 | | Hybrid | |
| 8 | SLC26A4 | c.1174A>T | Hybrid | |
| 9 | SLC26A4 | c.919-2A> | Homozygous mutations | |
| 10 | SLC26A4 | c.919-2A>G | Hybrid | |
| 11 | SLC26A4 | c.919-2A> | Hybrid | |
| 12 | SLC26A4 | c.919-2A>G | Hybrid |
1. Sample extraction:
human venous blood was collected and collected with EDTA anticoagulant tubes. 200. mu.L of peripheral blood was taken, genomic DNA was extracted using DNeasy blood and Tissue kit from QIAGEN, and the extracted DNA was quantified using Qubit and normalized to 5 ng/. mu.L.
2. And (3) fluorescent quantitative PCR detection:
the four-fold detection system of example 2 is adopted, the probes and primers designed in example 1 are used to respectively detect 16 deafness gene mutation sites of DNA extracted from 12 samples, and the corresponding detection sites comprise GJB2-c.176-191del16b, GJB2-c.35delG, GJB2-c.299-300delAT, GJB2-c.235delC, GJB2-512insAACG, GJB 3-c.53C > T, SLC A4-c.919-2A > G, SLC A4-c.2027T > A, SLC A4-c.2168A 4-c.1174A > T, SLC-A4-c.46G > A, SLC A4-c.1229C > 69526A 4-1707+ 5A > 5926-b.1178A > 4-c.4G > A, SLC A > 5926A > 5928-T, SLC C > 6959A 4-1707+ 5A > 56-b.1178A > 1978648-75G > 155 4-c > 1498627 and No. 20-b.5G > 1498627-b # C, MT.
3. And (4) analyzing results:
the results of 12 samples of deafness are shown in Table 5 below, in which white indicates wild type:
TABLE 5.12 samples of deafness
As shown in Table 5, the detection accuracy of SNP sites of deafness genes is as high as 100%. A considerable part of the hereditary hearing loss of Chinese population is caused by 16 high mutation sites of 4 common genes GJB2, GJB3, SLC26A4(PDS) and MT-RNR1(12S rRNA), and the sites have very high carrying rate in normal population and hearing impairment population. The detection accuracy of the invention reaches 100%, and the invention can be effectively applied to the gene detection of the neonatal deafness, so that the mutant patient can be intervened and treated in time, or the deafness-causing medicine can be avoided; meanwhile, the detection system can be used for detecting genes before marriage and before pregnancy, provides scientific judgment basis for prepotency, and carries out timely intervention and treatment on possible risks.
The invention carries out detection by SNP detection of deafness related susceptibility genes, but the method provided by the invention is not limited to the application of the deafness susceptibility genes, and other SNP typing detection extended by the method belongs to the protection scope of the patent.
The sample used in the practice of the present invention is leukocyte DNA, but is not limited thereto, and any other nucleic acid sample involved in the SNP typing detection associated with the present invention should be included in the scope of protection of the present patent.
In conclusion, the beneficial effects of the invention are as follows:
1. different SNP sites in the invention can use the same probe system, and compared with the Taqman method, the use of the probe (the synthesis price of the probe is high) is reduced, and the detection cost, especially the research and development cost, is greatly saved.
2. The invention can adopt 4 channels to detect 2 SNP sites simultaneously, and compared with the conventional 2 channels to detect 1 SNP site, the flux is doubled.
3. The invention adopts high-fidelity DNA polymerase with 3 ' to 5 ' exonuclease activity and a primer modified by 3 ' end sulfurated phosphoric acid, which can greatly reduce non-specific amplification, thereby accurately typing SNP loci.
4. The primer system of the invention contains 16 SNP sites of susceptibility genes related to deafness, basically covers the main SNP sites of the deafness susceptibility genes in Chinese population, and the detection method provided by the invention can be used for accurately typing the SNP related to the deafness genes.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
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Claims (10)
1. A probe system for gene SNP locus detection is characterized in that: the probe system at least comprises 2 probe pairs; each probe pair comprises an oligonucleotide sequence modified by a 5 'end fluorescent group and an oligonucleotide sequence modified by a corresponding complementary combined 3' end quenching group; the fluorophore for each probe pair is different; the Tm value of the oligonucleotide sequence modified by the 5 'end fluorescent group does not exceed the Tm value of the oligonucleotide sequence modified by the 3' end quenching group.
2. The probe system for genetic SNP site detection according to claim 1, characterized in that: the probe pair is selected from the following 4 probe pairs, wherein the probe pair 1 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.1 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 2;
the probe pair 2 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.3 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 4;
the probe pair 3 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.5 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 6;
the probe pair 4 comprises an oligonucleotide sequence modified by a 5 'end fluorescent group with a nucleotide sequence of SEQ ID NO.7 and an oligonucleotide sequence modified by a 3' end quenching group with a nucleotide sequence of SEQ ID NO. 8.
3. The probe system for genetic SNP site detection according to claim 1, characterized in that: the fluorophore modification comprises at least one of FAM, TET, JOE, HEX, CY3, CY5, ROX, and TAMRA; the quencher modification includes at least one of BHQ-1 and BHQ-2.
4. A reaction system for gene SNP locus detection is characterized in that: the reaction system comprises the probe system as claimed in any one of claims 1 to 3 and a primer set designed for the SNP site; each primer group comprises 1 wild type primer sequence and 1 mutant type primer sequence; the wild type primer and the mutant type primer both contain oligonucleotide sequences corresponding to probes with fluorescent groups in the probe pairs.
5. The reaction system for genetic SNP site detection according to claim 4, wherein: the 3' terminal base or adjacent base of the wild type primer sequence and the mutant type primer sequence is modified by phosphorothioate.
6. The reaction system for genetic SNP site detection according to claim 4, wherein: the reaction system also contains high-fidelity DNA polymerase; preferably, the high fidelity DNA polymerase includes at least one of Pfu polymerase and depvent polymerase.
7. A method for detecting gene SNP loci is characterized in that: the method comprises the following steps:
s1, constructing a reaction system according to any one of claims 4 to 6 for each SNP site;
and S2, carrying out PCR reaction to obtain a detection result.
8. The method for detecting SNP site of gene according to claim 7, wherein: the procedure for the PCR reaction was: 15min at 94 ℃; 94 ℃ for 20s, 60 ℃ for 60s, 10 cycles; collecting fluorescence at 94 ℃ for 20s and 55 ℃ for 60s, and collecting 35 cycles; fluorescence was collected at 30 ℃ for 60 s.
9. The application of the method for detecting SNP loci of genes according to claim 7 in detecting SNP loci related to deafness genes.
10. Use according to claim 9, characterized in that: the deafness gene related SNP loci comprise 16 SNP loci which are positioned at the correlation of four deafness genes, namely GJB2, GJB3, SLC26A4 and MT-RNR 1; the 16 SNP sites comprise GJB2-c.176-191del16b, GJB2-c.35delG, GJB2-c.299-300delAT, GJB2-c.235delC, GJB2-512insAACG, GJB3-c.538C > T, SLC26A4-c.919-2A > G, SLC26A4-c.2027T > A, SLC26A4-c.2168A > G, SLC26A 4-c.117A > T, SLC26A4-c.1226G > A, SLC26A 4-c.9C > T, SLC26A4-1707+5G > A, SLC26A 4-c.197G > C, MT-m.1555A > G and MT-m.1494T.
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