Detection method for number of multiple copies of SMN2 gene

Abstract

The invention discloses a detection method for the number of multiple copies of an SMN2 gene. The detection method comprises the following steps: S1, designing primers for specifically amplifying theSMN2 gene according to difference bases on the No.6 and No.7 introns of the SMN2 gene and NO.6 and NO.7 introns of an SMN1 gene, introducing mispairing bases at the antepenultimate bases of the 3' terminals of the primers for specifically amplifying the SMN2 gene, designing a specific detection probe SMN2-P aiming at the SMN2 gene, carrying out labeling at the 5' terminal and the 3' terminal of the probe, aiming at an RPP30 gene, designing the specific primers RPP30-F\RPP30-R and the probe RPP30-P, and carrying out labelling at the 5' terminal and the 3' terminal of the probe; S2, extracting sample DNA through peripheral blood; and S3, preparing a PCR reaction system, carrying out the PCR procedure, collecting the marks, and quantifying the copy number. The detection method is low in cost,and the accuracy in quantifying the detected copy number is high.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a quantitative PCR method for detecting the variation of SMN2 gene copy number. Background technique [0002] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, and the carrier frequency of the defective gene can be as high as 1 / 50. The key gene of SMA is motor neuron survival gene (Survival motorneuron, SMN). SMN is located on the long arm of chromosome 5 and contains two highly homologous copies, SMN1 and SMN2. Among them, SMN1 is the causative gene of SMA. About 95% to 98% of SMA patients are caused by homozygous deletion of SMN1. About 2% to 5% of patients have one copy of SMN1 missing and the other copy has a point mutation. There are 5 different bases between SMN1 and SMN2, distributed from intron 6 to exon 8. SMN1 can produce full-length motor neuron protein, and its homologous gene SMN2, due to the mutation on exon 7 (c.840C>T), causes th...

AI Technical Summary

Problems solved by technology

Single-strand conformational polymorphism analysis is based on the different exclusion resistances of single-stranded DNA with spatial conformation differences in polyacrylamide gels to detect mutations. This method is cumbersome and prone to false positives / negatives. undetectable

Denaturing high-performance liquid chromatography can be used to detect the difference in column elution between heteroduplexes and homoduplexes. It is highly automated and high-throughput. It can detect SMN1 and SMN2 gene copy numbers at the same time. It is widely used. The disadvantages are It is easily affected by temperature, eluent, etc., resulting in poor stability of the results, and requires PCR post-processing, which is easy to cause pollution

The high-resolution melting curve analysis method detects mutations based on the difference in the shape of the melting peak. The detection results of this method require manual interpretation, the accuracy is poor, and the quality of the template is high.

MLPA is an accurate and fast copy number detection method, but when there is a SNP in the probe binding region, the result will be inaccurate, which may cause false positives, and the operation is difficult, costly, and requires high purity of the sample

[0004] The market needs a low-cost, high-accuracy detection method for copy number quantification of multiple copies of the SMN2 gene. According to relevant literature and patent reports, qPCR can generally be used to detect low copy numbers of SMN1 and SMN2 genes, and 4 copies of the SMN2 gene The above resolution is low, but distinguishing high copy SMN2 is of great significance for the judgment of SMA disease type and guidance of clinical prognosis

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