OPRM1基因PCR产物和测序产物三种纯化方法的比较

摘要

Objective Looking for a quick,simple and effective purification method for PCR and sequencing product to establish stable DNA sequencing technology platform. Methods 50 PCR products and sequencing products of OPRM1 gene were purified by ethanl/sodium acetate,spin-column and SAP-Exonuclease I combined with BigDye Xterminator Purification Kit. Results The recovery rate of PCR products that was purified by SAP-Exonuclease I methods was significantly higher than by ethanl/sodium acetate and spin-column methods. Their recovery rates were 0. 86 ± 0. 01,0. 48 ± 0. 08, 0. 65 ± 0. 01 ( t= 32. 5,P<0. 001; t=86. L,P<0. 001). And the success rates of sequencing products that was purified by BigDye Xterminator Purification Kit was significantly higher than by ethanl/sodium acetate and spin-column. Their success rates were 100%, 62% and 84%(X2=23. 46, P< 0. 001 ;X2 =8. 70, P=0. 003). Conclusion The method of SAP-Exonuclease I combined with BigDye Xterminator Purification kit is a quick,easy and effective purification method.%目的 探讨一种快速、简便、效果好的OPRM1基因PCR产物和测序产物纯化方法,建立稳定的DNA测序技术平台.方法 采用乙醇/醋酸钠、过柱和SAP-Exonuclease Ⅰ结合BigDye Xterminator Purification Kit 3种纯化方法,对50份OPRM1基因PCR产物和测序产物进行纯化效果的比较.结果 SAP-Exonuclease Ⅰ法对PCR反应产物进行纯化的回收率显著高于乙醇/醋酸钠和过柱法,分别为0.86±0.01,0.48±0.08,0.65±0.01(t=32.5,P＜0.001；t=86.1,P＜0.001)；BigDye Xterminator Purification Kit法对测序产物进行纯化成功率显著高于乙醇/醋酸钠和过柱法,分别为100％,62％和84％(x2=23.46,P＜0.001;x2 =8.70,P=0.003).结论 SAP-Exonuclease Ⅰ结合BigDye Xterminator Purification Kit是一种快速、简便、效果好的测序纯化方法.