论文部分内容阅读
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。
Widespread use of DNA as a template for PCR methods are mainly CTAB and SDS 2, but cumbersome steps, time-consuming. According to Taq enzyme storage solution and PCR reaction buffer contained K + ions, Cl- ions and Tween-20 and other components, with 0.05mol / LKOH as alkaline lysis, 2% Tween-20 as stabilizer to prepare alkaline lysis solution to 0.05mol / L Tris-HCl and 2mmol / LEDTA were used as neutralization solution, and the PCR template was prepared by two steps of pyrolysis and neutralization. Using sugarcane leaves as materials, freshly prepared lysis solution containing KOH and Tween-20, the appropriate amount of leaves into a certain volume of lysis solution, heating and cracking, and then neutralized to form a lysis mixture. The pyrolysis mixture was directly used as a template and the endogenous gene of sugarcane was used as target gene. The PCR reaction was stable and reproducible with optimized KOH lysis concentration, template volume, pyrolysis time and the addition of appropriate amount of polymerase stabilizer. The representative monocotyledonous and dicotyledonous leaves were used as materials to verify the wide applicability of this method after repeated validation of multiple endogenous PCR reactions. The method prepares the PCR template in two steps without the DNA extraction process and uses less materials to perform the real-time PCR detection, thereby providing a simple, rapid and efficient analysis method for genetic analysis and molecular diagnosis.