Molecule by molecule PCR amplification of complex DNA mixtures for direct sequencing: An approach to in vitro cloning
Molecule by molecule PCR amplification of complex DNA mixtures for direct sequencing: An approach to in vitro cloning
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DOI：
10.1093/nar/24.11.2194
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发表时间：
1996-06-01
影响因子：
14.9
通讯作者：
Lukyanov, SA
中科院分区：
文献类型：
--
作者：
Lukyanov, KA;Matz, MV;Lukyanov, SA
研究由未知序列组成的复杂DNA样本的需要仍然促使研究人员使用体内克隆(在细菌、噬菌体和其他体内系统中克隆)作为使样本适合于筛选和测序的唯一方法。克隆的主要任务--单个分子的扩增--用聚合酶链式反应比较容易解决。它需要将接头连接到样品中的所有DNA末端，然后稀释样品以产生一个浓度，在该浓度下，用于扩增的体积包含大约一个分子，并用与接头相对应的引物进行聚合酶链式反应(‘克隆片段’)。然而，在这种情况下，扩增产物在两端具有相同的侧翼序列，因此不能使用这些侧翼作为引物退火点进行测序-两个文本将被同时阅读。我们已经开发了一种技术，虽然接近上述简单的方案，但确保最终的扩增产物在不同末端由不同的接头序列组成。这样的片段可以很容易地从两端进行测序。在我们的体外克隆过程中，我们将一对假双链接头(1)混合连接到样本中的所有DNA分子上。每个接头都是一对长度不均匀的寡核苷酸，在较长的寡核苷酸的3‘端相互互补(图1A)，形成一种结构，能够像正常的钝端一样参与连接。然而，只有较长的寡聚被连接，因为两个寡聚的5‘-端，包括较短的，没有磷酸基团。与连接的较长寡核苷酸互补的DNA拉伸在第一个PCR周期之前产生，此时较小的寡核苷酸已经脱离其结合位置，但较长的DNA分子仍保持其双链结构(事实上，它发生在样品在第一变性阶段之前加热时)。接头应该相当长(约40bp)，原因有两个：(I)能够用嵌套引物进行两轮聚合酶链式反应，已知这比一轮扩增更有效和特异；(Ii)抑制在连接过程中两端获得相同接头的分子的扩增。只有两个接头两侧不对称的分子才能被扩增，因为DNA链变性后，每条对称侧翼分子链的互补末端相互退火，在引物可以与之结合之前隐藏了引物的退火点。这一效果
The need to investigate a complex DNA sample consisting of unknown sequences still prompts the researcher to use in vivo cloning (cloning in bacteria, in phages and in other in vivo systems) as the only way to make the sample suitable for screening and sequencing. Here we demonstrate that it can be easily done by PCR, which saves a lot of effort.The main task of cloning—the amplification of individual molecules—is relatively easy to solve by PCR. It requires the ligation of adapter to all DNA ends in the sample, then dilution of the sample to produce a concentration at which the volume being taken for amplification contains about one molecule, and PCR (‘cloning takes’) with a primer which corresponds to the adapter. However, in this case the amplification products would have the same flanking sequence at both ends and therefore could not be sequenced using these flanks as primer annealing sites—two texts would be read simultaneously. We have developed a technique which, though being close to the aforementioned simple scheme, ensures that the final amplification product is flanked by different adapter sequences at the different ends. Such fragments can be sequenced easily from either of the ends. In our in vitro cloning procedure, we ligate a pair of pseudo double-stranded adapters (1) in a mix to all DNA molecules in the sample. Each adapter is a pair of oligonucleotides of uneven length complementary to each other at the 3′-end of the longer oligo (Fig. 1A), forming a structure that is able to participate in ligation as a normal blunt end. However, only the longer oligo is ligated, because the 5′-ends of both oligos, including the shorter one, lack phosphate groups. The DNA stretch complementary to the ligated longer oligo is produced before the first PCR cycle at temperatures when the smaller oligo already falls off its binding site, but longer DNA molecules still retain their double-stranded structure (in fact, it occurs while the sample is heated prior to the first denaturation stage). The adapters should be rather long (about 40 bp) for two reasons:(i) to make it possible to perform two rounds of PCR with nested primers, which is known to be much more powerful and specific than a one-round amplification;(ii) to suppress the amplification of molecules which had obtained identical adapters at both ends during ligation. Only molecules asymmetrically flanked by the two adapters can be amplified, because after the denaturation of DNA strands the complementary ends of each strand of symmetrically flanked molecules anneal to each other, hiding the primer annealing site before the primer can bind to it. This effect