Ligation: Joining DNA molecules tog

Ligation: Joining DNA molecules togetherThe final step in the construction of a recombinant DNA molecule is a joining togetherof the vector molecule and the DNA to be cloned (Figure 4.19). This process is referredto as ligation, and the enzyme that catalyses the reaction is called DNA ligase.4.3.1 The mode of action of DNA ligaseAll living cells produce DNA ligases, but the enzyme used in genetic engineering is usually purified from E. coli bacteria that have been infected with T4 phage. Within thecell, the enzyme carries out the very important function of repairing any discontinuitiesthat may arise in one of the strands of a double-stranded molecule (see Figure 4.4a).A discontinuity is quite simply a position where a phosphodiester bond between adjacent nucleotides is missing (contrast this with a nick, where one or more nucleotides areabsent). Although discontinuities may arise by a chance breakage of the cell’s DNAmolecules, they are also a natural result of processes such as DNA replication andrecombination. Ligases therefore play several vital roles in the cell.In the test tube, purified DNA ligases not only repair single-strand discontinuitiesbut also join together individual DNA molecules, or the two ends of the same molecule.The chemical reaction involved in ligating two molecules is exactly the same as discontinuity repair, except that two phosphodiester bonds must be made, one for each strand(Figure 4.20).4.3.2 Sticky ends increase the efficiency of ligationThe ligation reaction in Figure 4.20 shows two blunt-ended fragments being joinedtogether. Although this reaction can be carried out in the test tube, it is not very efficientbecause the ligase is unable to ‘catch hold’ of the molecule to be ligated and so mustwait for chance associations to bring the ends together. If possible, blunt end ligationshould be performed at high DNA concentrations, to increase the chances of the endsof the molecules coming together in the correct fashion.In contrast, the ligation of complementary sticky ends is much more efficient. Thisis because compatible sticky ends can base pair with one another by hydrogen bonding(Figure 4.21), forming a relatively stable structure for the enzyme to work on. If thephosphodiester bonds are not synthesized fairly quickly the sticky ends will fall apartagain. Nonetheless, these transient, base-paired structures increase the efficiency of ligation

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结果 (简体中文) 1: [复制]

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连接：将DNA分子连接在一起 构建重组DNA分子的最后一步是 将载体分子和要克隆的DNA 连接在一起（图4.19）。该过程称为 连接，而催化该反应的酶称为DNA连接酶。 4.3.1 DNA连接酶的作用方式 所有活细胞都产生DNA连接酶，但是通常从感染了T4噬菌体的大肠杆菌中纯化用于基因工程的酶。在 细胞内，该酶具有非常重要的功能，即修复 双链分子的一条链中可能出现的任何不连续性（参见图4.4a）。 不连续性非常简单，是在相邻核苷酸之间缺少磷酸二酯键的位置（将此与缺刻（一个或多个核苷酸 不存在的切口）进行对比）。尽管不连续性可能是由于细胞DNA 分子的偶然断裂而引起的，但它们也是DNA复制和 重组等过程的自然结果。因此，里加斯在细胞中起着至关重要的作用。 在试管中，纯化的DNA连接酶不仅可以修复单链不连续性 ，还可以将单个DNA分子或同一分子的两端连接在一起。 连接两个分子所涉及的化学反应与双键修复完全相同，不同之处在于必须进行两个磷酸二酯键的连接，每条链的一个 （图4.20）.4.3.2粘性末端提高了连接效率 图4.20中的连接反应显示了两个平末端片段连接 在一起。尽管此反应可以在试管中进行，但不是很有效， 因为连接酶无法“抓住”要连接的分子，因此必须 等待偶然的机会将末端连接在一起。如果可能， 应在高DNA浓度下进行平末端连接，以增加 分子末端以正确方式聚集在一起的机会。 相反，互补粘性末端的结扎效率更高。这 是因为兼容的粘性末端可以通过氢键彼此碱基配对 （图4.21），形成了一个相对稳定的结构，供酶作用。如果 磷酸二酯键不能很快合成，那么粘性末端将 再次破裂。但是，这些瞬时的碱基配对结构提高了连接效率。

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结果 (简体中文) 2:[复制]

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Ligation: Joining DNA molecules together The final step in the construction of a recombinant DNA molecule is a joining together of the vector molecule and the DNA to be cloned (Figure 4.19). This process is referred to as ligation, and the enzyme that catalyses the reaction is called DNA ligase. 4.3.1 The mode of action of DNA ligase All living cells produce DNA ligases, but the enzyme used in genetic engineering is usually purified from E. coli bacteria that have been infected with T4 phage. Within the cell, the enzyme carries out the very important function of repairing any discontinuities that may arise in one of the strands of a double-stranded molecule (see Figure 4.4a). A discontinuity is quite simply a position where a phosphodiester bond between adjacent nucleotides is missing (contrast this with a nick, where one or more nucleotides are absent). Although discontinuities may arise by a chance breakage of the cell’s DNA molecules, they are also a natural result of processes such as DNA replication and recombination. Ligases therefore play several vital roles in the cell. In the test tube, purified DNA ligases not only repair single-strand discontinuities but also join together individual DNA molecules, or the two ends of the same molecule. The chemical reaction involved in ligating two molecules is exactly the same as discontinuity repair, except that two phosphodiester bonds must be made, one for each strand (Figure 4.20).4.3.2 Sticky ends increase the efficiency of ligation The ligation reaction in Figure 4.20 shows two blunt-ended fragments being joined together. Although this reaction can be carried out in the test tube, it is not very efficient because the ligase is unable to ‘catch hold’ of the molecule to be ligated and so must wait for chance associations to bring the ends together. If possible, blunt end ligation should be performed at high DNA concentrations, to increase the chances of the ends of the molecules coming together in the correct fashion. In contrast, the ligation of complementary sticky ends is much more efficient. This is because compatible sticky ends can base pair with one another by hydrogen bonding (Figure 4.21), forming a relatively stable structure for the enzyme to work on. If the phosphodiester bonds are not synthesized fairly quickly the sticky ends will fall apart again. Nonetheless, these transient, base-paired structures increase the efficiency of ligation

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结果 (简体中文) 3:[复制]

复制成功！

连接：将DNA分子连接在一起 构建重组DNA分子的最后一步是连接在一起 载体分子和要克隆的DNA（图4.19）。此过程被引用 作为连接，而催化反应的酶称为DNA连接酶。 4.3.1 DNA连接酶的作用方式 所有活细胞都产生DNA连接酶，但用于基因工程的酶通常是从感染了T4噬菌体的大肠杆菌中纯化出来的。在 细胞，这种酶具有修复任何不连续性的重要功能 可能出现在双链分子的一条链中（见图4.4a）。 不连续性是一个很简单的位置，即adjacent核苷酸之间缺少一个磷酸二酯键（与此相反，缺口处有一个或多个核苷酸 缺席）。虽然细胞DNA的偶然断裂可能会引起间断 分子，它们也是DNA复制和 重组。因此，连接酶在细胞中起着重要的作用。 在试管中，纯化的DNA连接酶不仅修复单链不连续性 也可以把单个的DNA分子，或同一分子的两端连接起来。 连接两个分子所涉及的化学反应与discon连续修复完全相同，只是必须形成两个磷酸二酯键，每条链一个 （图4.20）4.3.2粘端增加结扎效率 图4.20中的结扎反应显示两个钝端片段连接在一起 一起。虽然这种反应可以在试管中进行，但不是很有效 因为连接酶不能“抓住”要连接的分子，所以必须 等待机会协会把结果结合起来。如果可能，钝端结扎 应该在高DNA浓度下进行，以增加结束的机会 分子以正确的方式聚集在一起。 相比之下，结扎互补的粘性末端更有效。这个 是因为相容的粘端可以通过氢键相互配对 （图4.21），形成一个相对稳定的结构供酶作用。如果 磷酸二酯键合成得不太快，粘端会脱落 再一次。尽管如此，这些瞬态的碱基对结构提高了发光效率

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