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Detection primers for gene mutation related to spinal muscular atrophy and its application

A spinal muscular atrophy and genetic technology, applied in the detection of spinal muscular atrophy-related gene mutations and genetic testing, can solve the problems of easy errors, high reagent costs, and inability to detect SMN1 gene copy number, etc., and achieve rapid detection results , low cost effect

Inactive Publication Date: 2018-01-26
陈万金
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These three techniques have their own disadvantages. The PCR-RFLP technique can only detect the homozygous deletion of the SMN1 gene, but cannot detect SMN1 The copy number of the gene; real-time PCR technology can only use a pair of internal reference genes to compare with the target gene, which is prone to errors; MLPA technology can design multiple pairs of internal reference genes for comparison at the same time, but the cost of reagents is high and time-consuming

Method used

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  • Detection primers for gene mutation related to spinal muscular atrophy and its application
  • Detection primers for gene mutation related to spinal muscular atrophy and its application
  • Detection primers for gene mutation related to spinal muscular atrophy and its application

Examples

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Embodiment 1

[0024] Example 1: Technical basis

[0025] The invention adopts the fluorescent short-segment multiplex PCR combined with the fragment analysis detection method to detect the mutation of the spinal muscular atrophy related gene. The fluorescent short-segment multiplex PCR method contains two or more pairs of fluorescently labeled primers in one PCR reaction system to detect multiple target sequences. At the same time, the gene analyzer is used to analyze the fragments of the amplified products, and the fluorescent signals of the corresponding fragments can be read, and the quantitative analysis can be carried out by obtaining the peak height and peak area of ​​the fragments.

Embodiment 2

[0026] Example 2: Primer Design

[0027] According to the exon 7 sequence of the SMN1 gene, the primers for specific amplification of the SMN1 gene were designed. Since the SMN1 gene and the SMN2 gene are highly homologous, with only 5 base differences, the 3' end of the upstream primer sequence of the SMN1 gene was specifically amplified. Contains the c.844 C>T difference site located in exon 7, the 3' end of the downstream primer sequence contains the g.27269A>C difference site located in intron 7, and the penultimate base at the 3' end of the downstream primer The mismatch is C, and the specific sequences are SEQ ID NO.1 and SEQ ID NO.2. This primer design can improve the specificity of PCR amplification of SMN1 gene. Three pairs of conserved sequences were selected as internal reference genes, and the specific primer sequences were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. The 5' end of the upstream sequence of all primers was labe...

Embodiment 3

[0028] Example 3: SMN1 Gene Fluorescent Short Fragment Multiplex PCR Detection

[0029]Reaction system: primer mixture 9 μl, 2×Buffer I (Takara, Japan) 12.5 μl, 25Mm MgCl 2 1 μl, 10mM dNTP (Takara Company, Japan) 1 μl, 5U / μl LaTaq enzyme (Takara Company, Japan) 0.25 μl, DNA 50ng.

[0030] PCR instrument: ABI Veriti (ThermoFisher Scientific, USA)

[0031] Reaction conditions: 96°C for 3 minutes; 96°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, 23 cycles; 72°C for 10 minutes.

[0032] Results: Analysis of 3730 results showed that the fragment size of 307nt was the SMN1 gene, and the fragment size of 209nt, 245nt, and 325nt were the positions of three pairs of internal reference genes.

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Abstract

The invention relates to detection primers for gene mutation related to spinal muscular atrophy and its application. The detection primers comprise a pair of primers specifically detecting the exon No.7 of a SMN1 gene, and three pairs of reference gene primers, wherein the nucleotide sequences of the pair of primers are SEQ ID No. 1 and SEQ ID No. 2, respectively, and the nucleotide sequences of the three pairs of reference gene primers are SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively. The above 4 pairs of primers are utilized for simultaneous fluorescent short-segment multiplex PCR detection of to-be-detected samples and normal samples; fluorescent signal data is collected; and whether mutation exists in the DNAs of the to-be-detected samples is analyzed. The invention also relates to a detection kit. The primers and kit provided by the invention are ingenious in design, simultaneously detect the SMN1 gene and a plurality of pairs of reference genes and compare the SMN1 gene with the plurality of pairs of reference genes, thereby more accurately reflecting the mutation of to-be-detected genes.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to the technical field of spinal muscular atrophy-related gene mutation detection, in particular to a spinal muscular atrophy-related gene detection method, a combination of related detection primers, a detection kit and related applications. Background technique [0002] Spinal muscular atrophy (SMA) is a relatively common genetic disease of the nervous system. It is an autosomal recessive genetic disease of limb muscle weakness and muscle atrophy caused by degeneration of the anterior horn of the spinal cord. The incidence rate of the disease is 1 / 6000 ~ 1 / 10000, the carrier frequency is 1 / 40 ~ 1 / 60. The causative gene of SMA is the motor neuron survival (Survival motor neuron, SMN) gene located on 5q13. Its main clinical manifestations are progressive and symmetrical limb weakness and muscle atrophy, with more proximal than distal, facial muscles and eyes The extrinsic muscles are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
Inventor 陈万金
Owner 陈万金
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