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目的介绍变性高效液相色谱(denaturinghighperformanceliquidchromatography,DHPLC)技术在儿童型脊髓性肌萎缩症(spinalmuscularatrophy,SMA)基因诊断中的应用。方法PCR扩增25名正常人、1份标准样品及25例SMA患者运动神经元生存基因(survivalmotorneuron,SMN)第7外显子及其侧翼区域,PCR产物变性、复性后直接上样于DHPLC系统。通过改变A、B缓冲液的比例来分离各种DNA成份。结果各种不同DNA成份以色谱峰的形式表现出来。23名正常标本呈现3个峰,依次为SMN1/SMN2异源双链峰、SMN2同源双链峰、SMN1同源双链峰。2名正常标本及1份标准品只有SMN1峰,表明缺失了SMN2。22例SMA患者只有SMN2峰,表明缺失了SMN1。另3例SMA患者呈现3个峰,表明无SMN1或SMN2缺失。结论DHPLC诊断SMA具有敏感、准确、快速、简便等优点。
Objective To introduce the application of denaturing high performance liquid chromatography (DHPLC) in gene diagnosis of children with spinal muscular atrophy (SMA). Methods The exon 7 and its flanking region of survival motoneuron (SMN) were amplified by PCR from 25 normal subjects, 1 standard specimen and 25 SMA patients. The PCR products were denatured and renaturated directly on DHPLC system. By changing the ratio of A, B buffer to separate various DNA components. As a result, various DNA components appear as chromatograms. 23 normal specimens showed three peaks, followed by SMN1 / SMN2 heteroduplex, SMN2 homoduplex and SMN1 homoduplex. There were only SMN1 peaks in two normal specimens and one standard, indicating that SMN2 was missing in only 22 of the SMA patients and only had an SMN2 peak, indicating the absence of SMN1. Three other SMA patients showed three peaks, indicating no deletion of SMN1 or SMN2. Conclusion DHPLC diagnosis of SMA is sensitive, accurate, fast, simple and so on.