用于检测脊髓性肌萎缩症基因（SMN1）缺失的新型改良版mCOP-PCR筛查系统

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

作者信息

Shinohara Masakazu, Ar Rochmah Mawaddah, Nakanishi Kenta, Harahap Nur Imma Fatimah, Niba Emma Tabe Eko, Saito Toshio, Saito Kayoko, Takeuchi Atsuko, Bouike Yoshihiro, Nishio Hisahide

机构信息

Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.

Division of Child Neurology, Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama, Japan.

出版信息

Kobe J Med Sci. 2017 Sep 7;63(2):E37-E40.

PMID:29434172

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5826017/

Abstract

BACKGROUND

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results.

AIM

To establish an improved version of the mCOP-PCR screening system without non-specific amplification.

METHODS

DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels.

RESULTS

No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system.

CONCLUSION

An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

摘要

背景

脊髓性肌萎缩症（SMA）是一种常见的常染色体隐性疾病，其特征为脊髓中的下运动神经元丧失。超过95%的SMA患者表现出纯合子生存运动神经元1（SMN1）缺失。我们之前基于改良的竞争性寡核苷酸引物PCR（mCOP-PCR）技术开发了一种用于筛查SMN1缺失的系统。然而，mCOP-PCR会出现非特异性扩增产物，这可能导致对筛查结果的错误解读。

目的

建立一个无非特异性扩增的改良版mCOP-PCR筛查系统。

方法

使用新版mCOP-PCR筛查系统对DNA样本进行检测。DNA样本已通过PCR-限制性片段长度多态性（PCR-RFLP）进行基因分型，显示存在或不存在SMN1外显子7。新的mCOP-PCR方法在mCOP-PCR步骤之前包含一个对该区域（包括一个SMN1特异性核苷酸）的靶向预扩增步骤。mCOP-PCR产物在琼脂糖凝胶上进行电泳。