摘要
广泛采用提取DNA作为PCR模板的方法主要有CTAB和SDS法2种,但步骤烦琐、耗时长。根据Taq酶储存液和PCR反应缓冲液所含K+离子、Cl-离子和Tween-20等成分,以0.05mol/LKOH为强碱裂解物、2%Tween-20为稳定剂配制碱裂解液,以0.05mol/L三羟甲基氨基甲烷盐酸(Tris-HCl)和2mmol/LEDTA为中和液,经加热裂解和中和两步制备PCR模板。以甘蔗叶片为材料,用新鲜配制含KOH和Tween-20的裂解液,将适量叶片放入一定体积的裂解液中加热裂解,再中和,形成裂解混合物。直接以裂解混合物为模板,甘蔗内源基因为靶基因,在优化KOH裂解浓度、模板体积、加热裂解时间以及添加适量聚合酶稳定剂的情况下,PCR反应稳定,重复性强。以有代表性的单子叶和双子叶植物叶片为材料,经过多种内源基因PCR反应的反复验证,证实了这种方法的广泛适用性。该方法通过两步制备PCR模板,无需DNA提取过程,使用材料少,可以实现活体PCR检测,为遗传分析和分子诊断提供简便、快速和高效的分析方法。
PCR templates are generally prepared by the method of CTAB or SDS, and these two methods are time-consuming and complicated in preparation procedure. A two-step handling procedure for preparation of PCR templates using alkali lysis was developed. Two kinds of solutions, namely lysis solution made from KOH and Tween-20, and neutralizing solution from Tris-HCl and EDTA were prepared based on components of known PCR reaction buffers and stock buffer of Taq enzyme. For the two-step procedure stock solutions of KOH and Tween-20 were mixed as lysis solution before use, and plant leaf tissues were placed in the lysis solution, heated and then neutralized with the neutralizing solution. These resulting template solutions worked well as templates in PCR reactions with presence of additives. An optimal protocol was set up based on optimization of volumes of templates, concentrations of KOH, and time span in lysis tested with sugarcane leaves and PCR thermo cycles of 5S rRNA-ITS. Other leaf samples were collected from monocotylous and dicotyledonous plants, respectively to test this procedure. A series of PCR reactions were tested, which proved this method was widely acceptable. The method for preparation of PCR templates is simple, rapid and efficient, offering a new approach for genetic analyses and molecular diagnosis.
出处
《热带作物学报》
CSCD
2010年第3期422-429,共8页
Chinese Journal of Tropical Crops
基金
国家863计划项目(2007AA100701)
福建省科技厅重点项目(2007I0036)
福建省外国专家局项目(20083500039)资助
关键词
KOH
碱裂解
中和
PCR模板
酶稳定剂
KOH
alkali lysis
neutralization
PCR templates
two-step handling.
