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分子生物学翻译

分子生物学翻译
分子生物学翻译

重组子的连接,转化与分析

DNA连接:为了将目标DNA片段插入到载体中,DNA分子共价连接的方法是至关重要的。DNA 连接酶执行这个功能;他们将修复双链DNA分子的其中一条链上的缺口(如图E2),提供5’末端一个磷酸基团。它们需要用激活酶试剂去激活被3'---OH攻击的磷酸基团;其中 E. co li 酶使用NAD+,更普遍使用的是来自T4噬菌体的酶使用A TP。连接酶对于密封一对退火粘性末端上的破损的磷酸二酯键是十分高效的(见图G2),事实上限制性内切酶反应的反面影响和T4连接酶甚至可以将一个平滑末端捆绑到另外一个末端上,尽管效率上相当低。

重组DNA分子:我们现在可以设想一个实验,实验内容是将一个包含感兴趣的(X)基因的DNA片段(目标DNA)插入到质粒载体中(如Fig.1)。目标DNA可能是一个从琼脂糖凝胶中分离出来的单一的片段(如Topic G3所示),又或者比如是来自基因组DNA的许多片段组成的混合物(图Topic Il)。如果目标DNA已经准备用EcoRI进行消化,那么片段将会结扎到被相同酶切割的载体DNA上。实际上,载体就应该只有一个位点进行相关酶的裂解,如果不是这样,正确的产品就只能结扎3个或者更多的片段而形成,这将十分没有效率。大多数产品来自于这个连接反应,并且结果取决于片段的相对浓度与自身条件,但是这个感兴趣的产品会以携带者目标片段的环状分子的形式插入到载体分子的E. co li位点,形成一个重组的分子(Fig. 1)。最初载体质粒的再生,通过线性载体单独的环化,这是一种能够鉴定出重组产品问题的竞争不良反应。一个方法是准备好目标DNA和载体同时用上一对独特的限制性内切酶,它们任何一个末端都要是不共存的粘性末端。这样结扎载体成为圆形的可能性就大大减小了。

碱性磷酸酶:如果不方便使用两种限制性酶,那么线性载体可以在使用限制性酶消化后再用碱性磷酸酶进行处理。碱性磷酸酶能够从DNA分子的5'末端移除磷酸基团。因此线性载体不能结扎成圆形状,因为没有磷酸盐能够对结扎反应起作用(见Fig. 2)。目标DNA插入结扎仍然可以进行,因为目前一个磷酸盐在每个切割位点只能结扎一条链(见Fig. 2)。将其他链上剩余的缺口将由细胞改造机制进行修复。

转化:通过结扎形成的其他质粒分子和重组混合物的组件必须相互隔离开来,并复制转移到宿主生物体。到目前为止,简单克隆实验中最常见的宿主细胞就是含有特定遗传特性的大肠杆菌菌株。一个明显的必要条件是,例如,它们必须是不能表达限制--修饰系统的。(见Topic G3)。研究发现,大肠杆菌细胞处理过的方案中包含的Ca2+变得容易被感染进而携带外源DNA,这是转化的一个过程。用Ca2+预处理细胞(有时也可以是其他的奇特的金属离子如Rb+和Mn2+),为了使它们能够携带DNA,这就是感受态细胞。大肠杆菌的转化,质粒分子的方案,或者是结扎反应形成的分子混合物,一个时期内是与感受态细胞的悬浮液相联系的,这样才能够携带上DNA。将DNA转移到细胞里的准确机制是难以捉摸的。该混合物在42℃下热休克1-2分钟。这促使了参与DNA修复的酶和其他细胞组件能够从转化过程的不正常状态中恢复过来,同时增强效率。细胞在生长培养基中培养,最后扩散到琼脂平板上,继续培养直到单个细菌菌落生长(见Fig. 3)。一个菌落里的所有细胞都来源于单一个体的分裂。所以,如果不包括自发突变,所有的细胞有相同的基因型(见Topic F1),这包括转换过程中出现的任何质粒(换言之,它们被克隆了)。

筛选:假如存在转化反应的所有感受态细胞可以在琼脂平板上生长,那么数千数百万的细菌群体就会产生。此外,转化是一个低效的过程;大部分产生的群体不包含质粒分子而且就算有了也不明显。选择含有质粒的克隆体是很有必要的。这大部分都是由质粒载体上的抗生素出现而提供的,比如β-1actamase基因(arnp r)赋予氨必西林抵抗性(见Topic G2)。如果转化细胞在包含氨必西林的平板上生长,那么就只有那些因为转化质粒的出现而进行β-1actamase基因表达的细胞会存活并

进行生长。我们能够进一步肯定,在氨必西林平板上形成的群体在转化后已经从单一含有一个完整的β-1actamase基因的细胞进而生长了。如果一个连接混合物用于转化,我们也不会知道这个阶段中,会含有一个包含目标片段的重组质粒的克隆体。

转化效率:提供准备的感受态细胞的质量能够侧得,这也决定了转化效率,使形成群体的数量更为明确,定义为输入DNA的数量每微克。DNA是一个纯化的质粒,很普遍的的,被作为载体使用于克隆实验中。天然转化规律中转化效率是从每微克103,这仅仅使转移一个完整质粒到新的宿主品种中成为可能,超过108每微克用来准备做感受态细胞的文库资料时要十分认真。大约l05每微克对于这里列出来的单个克隆实验是很充足的。

筛选转化子:如果在克隆实验中进行转化克隆,第一个条件是要知道哪个克隆体包含插入目标片段的重组质粒,已经确定了质粒来促进这一过程同时在Topic H1描述出来了。在许多情况下,将有必要确定数以千计的其他感兴趣的克隆,如筛选DNA库。这可能是所有过程中耗时最多的一部分。在一个简单的亚克隆实验中,实验设计可以最大限度地提高重组克隆的生产,例如碱性磷酸酶处理过的载体。因为这样,正常的筛选方法是准备要克隆的质粒DNA,然后用琼脂糖凝胶电泳分析。

转化子的生长与保存:单一菌落从改造板转移到培养液中,放置过夜直至达到稳定状态。培养液必须选择含有能够转化的抗生素的,以维持选择性质粒的存在。含有质粒的细菌在长期无选择增长中可能会丢失一些质粒,自带质粒的细菌则可能在竞争中意外失去质粒,因为这样能够使它们的复制需要更少的原料。质粒通过小量制备技术在培养液中做好准备。一般的做法是准备好要存储的每种培养基,将转化的细菌培养物分散于一定浓度的甘油溶液中冻存,形成冰冻晶体从而保护细胞。结果就是不用让相同的链或者质粒生长,而是从库存中一次又一次的取出同样的质粒。

凝胶电泳分析:重组质粒通常可以根据其相对大小简单区分开来,然后进一步的进行限制水解。图4显示了一个假设性的质粒分析的凝胶代表图。它表示相应的质粒载体和重组体的轨道。规模较大的重组质粒与未处理的质粒样品进行比较,不同的是包含超螺旋的和带有缺口的带,而且重组插入切除都是在大肠杆菌位点上消化。

片段定位:如果一个连接碎片反应要有一个载体和准备使用的单一限制性酶目标DNA,然后可以插入到载体环方向或者相连接。这可能是重要的,例如,如果目标插入含有编码基因的区域是被放在载体中启动子下游。片段的方向可以确定使用限制酶消化,这被削减不对称插入序列,然后同时在载体上的一个特定位点上。这在图1和4说明,使用插入序列削减酶贝(S)和HindlII(H),从而降解载体序列。预计这个模式会从两个方向,A和B的插入片段(H / S的轨道)。

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