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Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene

A single nucleotide polymorphism, gene technology, applied in the field of genetic engineering and gene diagnosis, can solve the problem of inability to judge the existence of heteroduplexes, and achieve the effect of high resolution

Inactive Publication Date: 2012-05-23
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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]

Method used

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  • Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene
  • Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene
  • Method for detecting single nucleotide polymorphism (SNP) of BANK1 (B-cell scaffold protein with ankyrin repeats 1) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Detection of polymorphism at rs10516487

[0052] 1. DNA extraction:

[0053] 2 ml of venous blood was collected using vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA), and DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany).

[0054] 2. Primer design:

[0055] Primers were designed using the 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.

[0056] 3. Probe design:

[0057] Primer Premier 5.0 software was used to design the probe. The probe of rs10516487 was 5'-GAAAAGAGAAATTCTCCAAGCGATATAACAGGATG-3' (SEQ ID NO 3). Probe extension....

Embodiment 2

[0065] Example 2 rs17266594 site polymorphism detection

[0066] 1. DNA extraction:

[0067] 2 ml of venous blood was collected using vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA), and DNA was extracted using the QIAamp DNA Extract Kit (Qiagen, Germany).

[0068] 2. Primer design:

[0069] Primers were designed using the 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'-CATTCCTCAGGCATCTTCTTCA-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.

[0070] 3. Probe design:

[0071] The probe was designed using Primer Premier 5.0 software, and the probe for rs17266594 was 5'-TAATAATTTAACCTGCTGATAGCATTGCAAATAT-3 (SEQ ID NO 6). The probe was synthe...

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Abstract

The invention which belongs to the fields of gene engineering and gene diagnosis provides a method for detecting SNP of a BANK1 gene. The method comprises the following steps: 1, designing a primer and a probe for amplifying an rs10516487 site or an rs17266594 site; 2, carrying out PCR amplification by utilizing the primer and the probe with sample DNA as a template, wherein concentrations of the upstream primer and the downstream primer in the primer are different, and the concentration of one is not less than five times the concentration of other one; and 3, adding a saturated dye, analyzing a melting degree curve, and determining the SNP. The method which can detect the polymorphism of the rs10516487 site and the rs17266594 site of the BANK1 gene in peripheral blood or a body fluid can be used to determine the attack possibility of autoimmtme diseases of systemic lupus erythematosus, rheumatoid arthritis, systemic chorionitis and the like. The method has the advantages of rapidness, simplicity, no pollution, and high detection result resolution. The invention also provides a corresponding detection kit.

Description

technical field [0001] The invention belongs to the field of genetic engineering and gene diagnosis, and relates to the detection and application of single nucleotide polymorphisms (SNP) at rs10516487 and rs17266594 sites of BANK 1 genes. Background technique [0002] 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 邹和建关明张心菊吴之源陈宇明
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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