Kesari Akanchha, Mukherjee Monisha, Mittal Balraj
Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India.
Indian J Biochem Biophys. 2003 Dec;40(6):439-41.
Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3' end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.
聚合酶链反应（PCR），随后进行限制性消化，被广泛用于脊髓性肌萎缩症（SMA）的分子诊断。在本研究中，我们采用了一种基于扩增阻滞突变系统-聚合酶链反应（ARMS-PCR）的改良策略，以开发一种快速且可靠的方法，用于SMA患者的突变检测和产前诊断。通过ARMS-PCR扩增存活运动神经元（SMN）基因第7外显子的端粒（SMN1）和着丝粒（SMN2）拷贝，使用针对SMN1和SMN2核苷酸序列的引物，其3'端具有外显子错配G（针对SMN1）和A（针对SMN2）。PCR产物在琼脂糖凝胶上进行分析。所有通过常规PCR-限制性片段长度多态性（PCR-RFLP）方法检测到SMN1基因第7外显子纯合缺失的患者，通过ARMS-PCR显示出相同的缺失状态。该方法在检测SMA患者的SMN1/SMN2状态时，PCR-RFLP和ARMS-PCR方法之间显示出100%的一致性。使用ARMS-PCR时，由于消化不完全导致的假象不是问题。该改良方案对于产前诊断同样具有特异性、快速且高度可靠。
