2.4 影响mtDNA 产率的因素
影响提取产率的最主要因素是线粒体的裂解程度, 试验过程中通过改变裂解缓冲液B 中蛋白酶K和SDS 浓度、裂解时间等条件, 未发现所提取的mtDNA 样品产率有显著变化。线粒体裂解时, 在裂解温度为37℃或50℃恒温裂解2 h, 发现对mtDNA 的产率影响不大, 平均每克新鲜的黄化苗分别可提取mtDNA 6.84 μg 和5.52 μg; 但在其他裂解条件不变的情况下, 先50℃恒温裂解1 h, 然后立即改变裂解温度至37℃再裂解1 h, 所得到的mtDNA 沉淀明显增多, 经测定平均每克新鲜的黄化苗可提取mtDNA 26.85μg(表2)。初步分析可能是温度的急剧改变引起线粒体膜破裂程度发生改变, 从而引起mtDNA 从线粒体膜内部更多地释放出来, 因此线粒体裂解更充分。
3 讨 论
为了获得高质量的mtDNA, 通常需要一系列操作步骤以去除蛋白质、次生代谢物、糖类小分子、核DNA 和RNA 等杂质, 用传统提取方法经过这些操作后所得到的mtDNA, 无论是完整度或提取产率方面均受到很大的影响。本研究以小麦黄化苗为材料, 通过差速离心、DnaseⅠ处理、线粒体裂解、酚 /氯仿抽提和RNase A 消化等主要步骤, 所提取的 mtDNA 结构完整、产率高, 不仅适合限制性酶切分析等对mtDNA样品需求量较大的研究, 而且适合对 mtDNA 纯度、完整度要求高的PCR 扩增。与传统的提取方法[10~12]相比, 本方法所提取mtDNA 不但纯度高、无降解、电泳谱带清晰, 而且具有以下优点; (1)在提取过程中, 用DNaseⅠ和RNase A 进行消化, 可排除mtDNA 中的核DNA 和RNA, 能保证后续分子操作的可靠性; (2)在线粒体裂解过程中, 通过调整裂解温度(先50℃裂解1 h, 再37℃裂解 1 h), 从而大大提高了mtDNA 的产率。此外, 用本方法从二角山羊草的成熟叶片中提取mtDNA, 也获得了很好的效果。
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