CN102465170A - A method for detecting single nucleotide polymorphism of BANK1 gene - Google Patents

Abstract

The invention belongs to the field of genetic engineering and gene diagnosis, and provides a method for detecting single nucleotide polymorphism of BANK1 gene, which comprises the following steps: firstly, designing primers and probes for amplifying rs10516487 or rs17266594 sites; then, taking the sample DNA as a template, and carrying out PCR amplification by using the primer and the probe; the concentration of the upstream primer and the concentration of the downstream primer in the primers are different, and one primer is not less than 5 times of the other primer; and finally, adding a saturated dye, analyzing a melting degree curve and determining the single nucleotide polymorphism. The method can detect the polymorphism of BANK1 genes rs10516487 and rs17266594 in peripheral blood or body fluid so as to judge the possibility of suffering from autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic scleroderma and the like. The method is rapid, simple, pollution-free and high in resolution of detection results. The invention also provides a corresponding detection kit.

Description

A method for detecting single nucleotide polymorphism of BANK1 gene

technical field

The invention belongs to the field of genetic engineering and genetic diagnosis, and relates to the detection and application of single nucleotide polymorphisms (SNP) at rs10516487 and rs17266594 sites of BANK 1 genes.

Background technique

Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple systems, and there are multiple autoantibodies in patients. The disease is more common in women and can cause damage to the skin, lungs, blood vessels and nervous system of the human body. Data show that if there is a family history of SLE, the incidence of SLE is dozens of times that of the general population. SLE is also related to race and ethnicity. For example, the incidence rate of black women in the United States is much lower than that of the general population in Europe and America, while in China, the incidence rate of SLE is higher than that in Europe and the United States. It can be seen that this disease is related to genetics and environment. At present, it is believed that the combination of factors such as genetics, hormones, and environment may cause immune regulation dysfunction, deposition of antigen, antibody, and complement complexes, and result in local or systemic tissue or organ damage. SLE patients are often accompanied by significant abnormal activation of T and B lymphocytes. B lymphocytes, as classic immune effector cells, play an important role in the process of SLE autoimmunity. the

Ankyrin repeat sequence 1 on B cell scaffold protein, referred to as BANK 1 (B-cell scaffold protein with ankyrin repeats 1). Scaffolding proteins are proteins that play an important role in regulating signal transduction by assisting protein kinases and phosphatases in binding to their respective substrates. Ankyrin repeat (ANK) is a sequence motif widely utilized in organisms. The ANK domain mediates protein-protein interactions, and it can bind to a variety of ligands to achieve complex biological functions. The B cell-specific scaffolding protein encoded by the BANK 1 gene plays an important role in the B cell receptor-induced intracellular calcium ion mobilization. It also promotes Lyn (a membrane-bound protein tyrosine kinase mainly expressed in hematopoietic cells)-mediated tyrosine phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R). IP3R is a chemically gated calcium release channel. Calcium ions are released from the endoplasmic reticulum into the cytoplasm through IP3R channels. The activation of IP3R can lead to the release of Ca ²⁺ in the endoplasmic reticulum or sarcoplasmic reticulum and the increase of Ca ²⁺ concentration in the cytoplasm. Subsequently, the B cell antigen receptor signaling pathway is activated through multiple signaling pathways such as NFAT, leading to the loss of B cell immune tolerance and abnormal increase in B cell reactivity.

In 2008, Kozyrev et al ^[1] first published a report on the relationship between BANK 1 and SLE susceptibility. Their research found that BANK 1 is a susceptibility gene for SLE, and two SNPs on BANK 1, rs10516487 and rs17266594, are closely related to the pathogenesis of SLE. This finding was also confirmed by Chang et al ^{. [2]} 's survey of the population in Hong Kong, China.

rs10516487 is located in the key region linking IP3R ^[1] , which is a non-synonymous alternative SNP site. Synonymous substitution only changes the codon but does not change the amino acid sequence of the translated protein, and it is generally believed that it does not affect protein structure and function. Codon variations that are not synonymous substitutions can lead to changes in the encoded amino acids, thereby affecting the physiological function of the protein. This variation is often the direct cause of changes in biological traits. The 61st amino acid of rs10516487 is changed from arginine to histidine.

Studies have found that ^[1] , BANK1 has two isoforms, one long and one short. The short subtype is called △2 subtype, which means that the exon 2 of the BANK1 gene is completely deleted, resulting in the lack of the IP3R binding region in the protein produced by its translation. rs17266594 is just located at the branch point of this sequence (Figure 1), and its T allele homozygote has a very typical branch point sequence (YNYTGAYYN) (Y: pyrimidine, N represents any nucleotide). The branch point sequence is a conserved sequence located near the 3' end of nuclear mRNA introns and class II introns. Intronic sequence branch points, intronic 5'-end splice junctions (donor site) and 3'-end splice junctions (acceptor site) are required for correct splicing of pre-mRNA. Therefore, the polymorphism of rs17266594 is directly related to the splicing efficiency of the Δ2 subtype of BANK 1 gene.

The polymorphisms of rs10516487 and rs17266594 may affect the function of the BANK 1 gene, thereby promoting the continuous release of signals from the B cell receptor through a series of reactions, resulting in excessive activation of B cells.

Currently, common detection methods for screening known SNPs include restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), allele-specific oligonucleotide fragment analysis (ASO), denaturing gradient Gel electrophoresis (DGGE), Taqman probe method, gene chip, sequencing and other technologies. However, each has its own disadvantages. For example, RFLP can only detect SNPs with enzyme cleavage sites, but cannot detect SNPs without enzyme cleavage sites; RFLP, SSCP, ASO, and DGGE all use electrophoresis methods, which have limited sensitivity and pollute the environment; Acupuncture is expensive; gene chips are not suitable for the detection of multiple samples and a small number of SNP sites; although sequencing is the gold standard for nucleotide detection, it is expensive and takes a long time, so it is not suitable for disease association analysis of large samples.

SNP detection using high-resolution melting method (High-resolution melting, HRM) is a new method introduced in recent years. It judges the nature of the product based on the melting temperature (Tm). The melting temperature is determined by the GC content, length, sequence, and heterozygosity of the PCR product. Combining a new type of saturating dye (such as SYTO 9) and a high-resolution PCR instrument (such as Rotor-Gene 6000), even if there is only one base change, it can be reflected from the melting temperature ^[3] . Only one dye is needed for the detection of any target gene. However, if there are multiple mutations in a sample at the same time, it cannot be judged by the above method alone. Or when the purity of the DNA sample is not high and primer dimers appear in PCR amplification, it will change the melting temperature detected by HRM, thereby affecting the judgment of the result. Therefore, although the Taqman probe method is more expensive and is subject to various limitations such as each fluorescent probe can only target one detection target, it is still widely used clinically ^[4] . For the detection of large-scale samples, in order to reduce the cost, an analytical method that combines the specificity of probes and the universality of dyes is urgently needed. Unlabeled probe high resolution melting (unlabeled probe high resolution melting, unlabeled probe HRM) fully meets these requirements.

The unlabeled probe method is further improved on the basis of PCR: 3'-end blocked unlabeled probe, dye and a pair of primers with inconsistent concentrations. SYBR Green I has been used as a classic dye in PCR for more than ten years. However, since this is an unsaturated dye, it cannot completely fill the minor groove of the DNA double strand, and the dissociated SYBR Green I during the melting of the DNA will recombine into the unmelted double strand, resulting in distortion of the results. Excess SYBR Green I also inhibits PCR amplification. In the unlabeled probe method, the concentration of SYBR Green I suitable for PCR amplification can only detect products with higher melting temperatures, and cannot determine the existence of heteroduplexes ^[5] .

references:

1. Kozyrev SV, Abelson AK, Wojcik J, et al. Functional variants in the B-cell gene BANK1 are associated with systemic lupus erythematosus. Nat genet, 2008, 40: 211-216.

2. Chang YK, Yang W, Zhao M, et al. Association of BANK1 and TNFSF4 with systemic lupus erythematosus in Hong Kong Chinese. Genes Immun, 2009, 10: 414-420.

3. Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics, 2007, 8: 597-608.

4. Erali M, Palais R, Wittwer C. SNP genotyping by unlabeled probe melting Analysis. Methods Mol Biol, 2008, 429:199-206.

5. Zhou L, Myers AN, Vandersteen JG, et al. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem, 2004, 50, 1328-1335.

Contents of the invention

The purpose of the present invention is to provide a simple method for detecting the polymorphism of BANK1 gene. The method is fast, simple, and non-polluting, and can detect various specimens, including serum (serum) and various body fluids.

Another object of the present invention is to provide a simple kit for detecting polymorphism of BANK1 gene.

the

In the unlabeled probe HRM, the unlabeled probe in the asymmetric PCR reaction will not be amplified under the action of the polymerase because the 3' end has been blocked. Relying on the upstream and downstream primers, a large number of paired target fragments can be amplified. When one primer is exhausted, the other excess primer will continue to amplify DNA single strands, and free probes will bind to these single strands. The probe binds to the single-stranded product amplified by asymmetric PCR, and the saturating dye binds to the duplex formed by the probe/product and product/product combination. Saturating dyes (such as SYTO 9, LC Green) have stronger DNA binding ability and low inhibition, which are completely suitable for the detection of single base changes.

If the product contains different alleles, the melting temperature of the duplex formed with the probe will show a significant difference. The same goes for mutual pairings between products. After analysis by the software that comes with the high-resolution instrument, different genotypes can be clearly displayed.

In the negative derivative plot of fluorescence (-dF/dT), there will be at least 2 melting peaks for each sample, one is the melting peak of the probe/product with a lower melting temperature, and the other is the product with a higher melting temperature / Melting peak of the product. If the amplified fragment contains other unknown base mutations, in addition to the above two, a third melting peak will appear.

In the melting peak of the probe/product with a lower melting temperature, if the single-stranded product amplified from the template DNA completely matches the probe, the melting temperature will be higher due to the need for higher energy to melt the double-strand ; If the SNP site in the template DNA to be tested does not completely match the probe, only low energy can be used to open the double-stranded structure, so the melting temperature is low; if the template DNA is heterozygous, its melting curve will be simultaneously It has the characteristics of the above two melting peaks.

In the melting peak of the product/product with a higher melting temperature, when there is no other base change in the amplified fragment except the site to be detected, the melting peak of the product/product is consistent with the melting peak of the probe/product; when When the amplified fragment also contains base changes at other sites, the shape of the product/melting peak of the product will change, suggesting that there are other base changes. Since the difference between product/product melting peaks between different bases is generally <0.5°C, it is easier to observe such base changes after converting the data into a normal view by software for partial zoom-in. In the double helix structure of DNA, G-C base pairs have 3 pairs of hydrogen bonds, and A-T base pairs have 2 pairs of hydrogen bonds. Therefore, it takes more energy to destroy the hydrogen bonds between G-C than A-T hydrogen bonds. Therefore, the CC/GG gene The melting temperature of the genotype is higher than that of the TT/AA genotype, while the heterozygote has a lower melting temperature due to weak combination. the

By combining the results of the fluorescence derivative graph and the normal view, the genotype of the polymorphic site of the BANK1 gene can be accurately judged.

the

The invention provides a method for detecting the single nucleotide polymorphism of the BANK 1 gene, which method comprises in turn: designing primers and probes for amplifying the sequence of the single nucleotide polymorphism site; using sample DNA as a template, PCR amplification is carried out by using the above primers and probes; saturation dyes are added; melting curves are analyzed to determine single nucleotide polymorphisms. Probes are designed according to the sequence of single nucleotide polymorphism sites. Primers were designed according to the principle of PCR and determined by subsequent PCR results.

Among the primers for PCR amplification, the concentrations of the upstream primer and the downstream primer are different, and the concentration of one is not less than 5 times that of the other. For example, the concentration of the downstream primer is 10 times, 15 times, 20 times, 25 times, 30 times, etc. of the upstream primer.

Said sample is blood component or body fluid.

The saturated dye is SYTO 9 or LC Green (commercially available, such as Invitrogen, USA).

The probe concentration is not less than the concentration difference of the two primers. The probe concentration can also be slightly less than the difference between the concentrations of the upstream and downstream primers, or the probe concentration can be equal to the one with the higher concentration.

In one embodiment of the present invention, the site of the single nucleotide polymorphism is rs10516487. The primer sequence is shown in SEQ ID NO 1 and SEQ ID NO 2, and the probe sequence is shown in SEQ ID NO 3.

In another embodiment of the present invention, the site of the single nucleotide polymorphism is rs17266594. The primer sequence is shown in SEQ ID NO 4 and SEQ ID NO 5, and the probe sequence is shown in SEQ ID NO 6.

Specifically, after the DNA is extracted from the sample in this method, the required DNA fragments are amplified by asymmetric polymerase chain reaction (PCR) with the addition of unlabeled probes, and saturated fluorescent dyes are added. Detect its fluorescence change. Each sample has 2 melting peaks, the melting peak with a lower temperature is used to judge the base change within the coverage of the probe, and the melting peak with a higher temperature is used to judge the base change in the entire target fragment.

1. DNA extraction from plasma.

2. Design primers.

3. Design a probe for the site to be tested, without fluorescent labeling, and block the 3' end with C3.

4. Add fluorescent dyes, such as SYTO 9, for asymmetric PCR amplification. After the end, the melting temperature is detected on a high-resolution PCR instrument, and the change of the fluorescent signal of the sample is analyzed when the temperature drops.

5. Analyze the rs10516487 and rs17266594 polymorphisms of the BANK1 gene in the sample to be tested.

Correspondingly, the present invention provides a kit for detecting the single nucleotide polymorphism of the BANK 1 gene, which includes primers and probes for amplifying the sequence of the single nucleotide polymorphism site. Saturation dyes, buffers, etc. may also be included.

The invented method for detecting the single nucleotide polymorphism of the BANK 1 gene can be used to predict the susceptibility of suffering from systemic lupus erythematosus, rheumatoid arthritis or systemic scleroderma.

Both BANK1 gene rs10516487 and rs17266594 loci are susceptibility loci for SLE, and the probability of SLE in people carrying the CT haplotype is 1.6 times that of normal people, which can be used to assess the risk of individuals suffering from the disease. the

The Δ2 subtype of BANK1 means that exon 2 of the BANK1 gene is completely deleted, and the translated protein lacks the IP3R binding region. rs17266594 is just at the branch point of this sequence of BANK1. Therefore, the polymorphism of rs17266594 is directly related to the splicing efficiency of BANK 1 gene and the function of translational protein. rs10516487 is located in the key region connecting IP3R, which is a non-synonymous alternative SNP site. The polymorphisms of rs10516487 and rs17266594 will affect the function of the BANK 1 gene, thereby promoting the continuous release of signals from the B cell receptor through a series of reactions, resulting in excessive activation of B cells.

the

The invention provides a method for detecting the single nucleotide polymorphism of the BANK1 gene, which can be used to detect the rs10516487 and rs17266594 polymorphisms of the BANK1 gene in peripheral blood or body fluid. To determine the possibility of suffering from systemic lupus erythematosus, rheumatoid arthritis, systemic scleroderma and other autoimmune diseases. The operation of this method has the characteristics of fast, simple and pollution-free, and the detection result has high resolution, and can detect various specimens, including serum (serum) and various body fluids. The invention also provides a corresponding detection kit.

Description of drawings

Figure 1: Schematic diagram of rs10516487 and rs17266594 loci. rs10516487 is a non-synonymous substitution SNP site, which leads to the change of amino acid 61 from arginine to histidine; rs17266594 is located at the branch point of the sequence and has a typical sequence of branch points.

Figure 2: Negative fluorescence derivative plot (-dF/dT) of the rs10516487 locus. On the left is the melting peak of the probe/product with a lower melting temperature, and on the right is the melting peak of the product/product with a higher melting temperature. The red line represents the CC genotype, the blue line represents the CT genotype, and the yellow line represents the TT genotype.

Figure 3: Generic view of the product/product melting curve for the rs10516487 site. The red line represents the CC genotype, the blue line represents the CT genotype, and the yellow line represents the TT genotype.

Figure 4: Negative fluorescence derivative plot (-dF/dT) of the rs17266594 locus. On the left is the melting peak of the probe/product with a lower melting temperature, and on the right is the melting peak of the product/product with a higher melting temperature. The red line represents the TT genotype, the blue line represents the TC genotype, and the yellow line represents the CC genotype.

Figure 5: Generic view of the product/product melting curve for the rs17266594 site. The red line represents the TT genotype, the blue line represents the TC genotype, and the yellow line represents the CC genotype.

Detailed ways

Example 1: Detection of polymorphism at rs10516487

1. DNA extraction:

2 ml of venous blood was collected using ethylenediaminetetraacetic acid (EDTA) anticoagulated vacuum blood collection tubes, and DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany) kit.

2. Primer design:

Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/), and after manual screening and PCR experiment verification, primers were obtained. The upstream primer F of rs10516487: 5'- ACATTTGTAAGACGTTAAGTTCAGCA-3' (SEQ ID NO 1), the downstream primer R: 5'-ATGATATATGAAGAAGATGCTGAGGA-3' (SEQ ID NO 2), the length of the amplified fragment is 150 bp. Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

3. Probe design:

The probe was designed using Primer Premier 5.0 software. The probe of rs10516487 was 5'-GAAAAGAGAAATTCTCCAAGCGATATAACAGGATG-3' (SEQ ID NO 3). Probe extension.

4. Sample testing:

l TaKaRa Taq HS（5 U/ μl），2 l 10 × PCR Buffer (-) (Mg²⁺ Free)，1.2 

l MgCl₂（25 mM），1.6 

l dNTP Mixture（各2.5 mM），上游引物F 1 

l（1

M），下游引物R 1 

l（10

M），探针1 

l（10

M），加水至总体积19.3 

l，然后加入0.7 

l样本DNA。PCR扩增条件：95℃ 2 min，94℃ 30 s、58℃ 30 s、72℃ 30 s，50个循环后，72℃ 5 min延伸。使用Eppendorf personal 基因扩增仪（Eppendorf公司，德国）扩增后加入0.7 

l SYTO 9 染料（50 

M）（Invitrogen公司，美国），再将样品管转移到Rotor-Gene 6000（Mortlake Corbett Research公司，澳大利亚），进行高分辨率熔解分析。条件为：95°C 2 min，25°C 2 min预处理后，熔解温度从55°C升至88°C，每升高0.2 °C采集一次数据，获得高分辨率熔解曲线图。 PCR amplification was performed using TaKaRa PCR kit (Takara Bio, Japan). The reaction system includes: 0.1

l TaKaRa Taq HS (5 U/μl), 2 l 10 × PCR Buffer (-) (Mg ²⁺ Free), 1.2

l MgCl ₂ (25 mM), 1.6

l dNTP Mixture (2.5 mM each), upstream primer F 1

l (1

M), downstream primer R 1

l (10

M), probe 1

l (10

M), add water to a total volume of 19.3

l, then add 0.7

l Sample DNA. PCR amplification conditions: 95°C for 2 min, 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, after 50 cycles, 72°C for 5 min extension. Add 0.7

l SYTO 9 dye (50

M) (Invitrogen, USA), and then transfer the sample tubes to a Rotor-Gene 6000 (Mortlake Corbett Research, Australia) for high-resolution melting analysis. The conditions were: 95°C for 2 min, 25°C for 2 min after pretreatment, the melting temperature increased from 55°C to 88°C, and the data were collected every 0.2 °C increase to obtain a high-resolution melting curve.

5. Polymorphism analysis

On the plot of the negative derivative of fluorescence (-dF/dT), on the left is the melting peak of the lower melting probe/product. If the rs10516487 base of the template DNA is cytosine (C), the amplified single-stranded product will completely match the probe, the melting temperature will be higher, and the melting peak will be to the right; if the base of the template DNA is Thymine (T), the single strand cannot completely match the probe, the melting temperature is low, and the melting peak is left; It will have the characteristics of the above two melting peaks at the same time (Figure 2).

On the right is the melting peak of the higher melting temperature product/product. The data is converted into a normal view through the software, and the melting curve of the product/product is partially enlarged to observe the base change. The Tm of the CC genotype at the rs10516487 locus is the highest, located on the right, followed by the TT genotype, while the CT genotype has the lowest Tm due to weak binding (Figure 3). the

By combining the results of the fluorescence derivative map and the normal view, the genotype of the rs10516487 site of the BANK1 gene can be accurately determined.

the

Example 2 rs17266594 site polymorphism detection

1. DNA extraction:

2 ml of venous blood was collected using ethylenediaminetetraacetic acid (EDTA) anticoagulated vacuum blood collection tubes, and DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany) kit.

2. Primer design:

Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/), and after manual screening and PCR experiment verification, primers were obtained. The upstream primer F of rs17266594: 5'-AGGACTTTCATAGAGTTTTTCTCTGG-3' (SEQ ID NO 4), the downstream primer R: 5'- CATTCCTCAGCATCTTCTTCA-3' (SEQ ID NO 5), the length of the amplified fragment is 185 bp. Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

3. Probe design:

The probe was designed using Primer Premier 5.0 software, and the probe of rs17266594 was 5'-TAATAATTTAACCTGCTGATAGCATTGCAAATAT-3 (SEQ ID NO 6). The probe was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the 3' end was blocked by C3 to prevent the probe from extending.

4. Sample testing:

l TaKaRa Taq HS（5 U/ μl），2 

l 10 × PCR Buffer (-) (Mg²⁺ Free)，1.2 

l MgCl₂（25 mM），1.6 

l dNTP Mixture（各2.5 mM），上游引物F 1 

l（0.67

M），下游引物R 1 l（10

M），探针1.5 

l（10

M），加水至总体积19.3 

l，然后加入0.7 

l样本DNA。PCR扩增条件：95℃ 2 min，94℃ 30 s、58℃ 30 s、72℃ 30 s，50个循环后，72℃ 5 min延伸。使用Eppendorf personal 基因扩增仪（Eppendorf公司，德国）扩增后加入0.7 

l SYTO 9 染料（50 

M）（Invitrogen公司，美国），再将样品管转移到Rotor-Gene 6000（Mortlake Corbett Research公司，澳大利亚），进行高分辨率熔解分析。条件为：95°C 2 min，40°C 2 min预处理后，熔解温度从57°C升至83°C，每升高0.2 °C采集一次数据，获得高分辨率熔解曲线图。 PCR amplification was performed using TaKaRa PCR kit (Takara Bio, Japan). The reaction system includes: 0.1

l TaKaRa Taq HS (5 U/μl), 2

l 10 × PCR Buffer (-) (Mg ²⁺ Free), 1.2

l MgCl ₂ (25 mM), 1.6

l dNTP Mixture (2.5 mM each), upstream primer F 1

l (0.67

M), downstream primer R 1 l (10

M), probe 1.5

l (10

M), add water to a total volume of 19.3

l, then add 0.7

l Sample DNA. PCR amplification conditions: 95°C for 2 min, 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, after 50 cycles, 72°C for 5 min extension. Add 0.7

l SYTO 9 dye (50

M) (Invitrogen, USA), and then transfer the sample tubes to a Rotor-Gene 6000 (Mortlake Corbett Research, Australia) for high-resolution melting analysis. The conditions were: 95°C for 2 min, 40°C for 2 min after pretreatment, the melting temperature increased from 57°C to 83°C, and data was collected every 0.2 °C increase to obtain a high-resolution melting curve.

5. Polymorphism analysis

On the plot of the negative derivative of fluorescence (-dF/dT), on the left is the melting peak of the lower melting probe/product. If the rs17266594 base of the template DNA is thymine (T), the amplified single-stranded product matches the probe completely, the melting temperature is higher, and the melting peak is to the right; if the base of the template DNA is Cytosine (C), the single strand cannot be completely matched with the probe, the melting temperature is low, and the melting peak is to the left; if the template DNA is heterozygous, part of the base at this site is T, and part of it is C, the melting curve It will have the characteristics of the above two melting peaks at the same time (Figure 4).

On the right is the melting peak of the higher melting temperature product/product. The data is converted into a normal view through the software, and the melting curve of the product/product is partially enlarged to observe the base change. The Tm of the CC genotype at the rs17266594 locus is the highest, located on the right, followed by the TT genotype, and the Tm of the TC genotype is the lowest due to weak binding (Figure 5). the

By combining the results of the fluorescence derivative graph and the normal view, the genotype of the rs17266594 locus of the BANK1 gene can be accurately determined.

Claims (10)

1. a method for detecting BANK 1 gene single nucleotide polymorphism, is characterized in that, the method comprises successively: Design primers and probes for amplifying the sequence of a single nucleotide polymorphism site; the single nucleotide polymorphism site is rs10516487 or rs17266594; Using the sample DNA as a template, use the above primers and probes for PCR amplification; the concentration of the upstream primer and downstream primer in the primers is different, and one is not less than 5 times the concentration of the other; add saturating dyes; Melting curves were analyzed to identify SNPs. 2. The method of claim 1, wherein the sample is a blood component or a body fluid. 3. the method for claim 1, is characterized in that, described saturation dyestuff is SYTO 9 or LC Green. 4. The method according to claim 1, wherein the concentration of the probe is not less than the difference between the concentrations of the two primers. 5. The method of claim 1, wherein the site of the single nucleotide polymorphism is rs10516487. 6. The method according to claim 5, wherein said primer sequence is as shown in SEQ ID NO 1 and SEQ ID NO 2, and said probe sequence is as shown in SEQ ID NO 3. 7. The method of claim 1, wherein the site of the single nucleotide polymorphism is rs17266594. 8. The method according to claim 7, wherein said primer sequence is as shown in SEQ ID NO 4 and SEQ ID NO 5, and said probe sequence is as shown in SEQ ID NO 6. 9. A kit for detecting the single nucleotide polymorphism of the BANK 1 gene, characterized in that the kit includes primers and probes for amplifying the sequence of the single nucleotide polymorphism site rs10516487 or rs17266594. 10. the application of the method described in claim 1 is characterized in that, utilizes the detection result of BANK 1 gene SNP to predict the susceptibility of suffering from systemic lupus erythematosus, rheumatoid arthritis or systemic scleroderma. emotional.

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