RECURSIVE PCR - A NOVEL TECHNIQUE FOR TOTAL GENE SYNTHESIS
RECURSIVE PCR - A NOVEL TECHNIQUE FOR TOTAL GENE SYNTHESIS
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DOI：
10.1093/protein/5.8.827
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发表时间：
1992-12-01
期刊：
影响因子：
--
通讯作者：
PEARL, LH
中科院分区：
文献类型：
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作者：
PRODROMOU, C;PEARL, LH
在涉及突变和表达来自重组DNA的蛋白质的蛋白质工程应用中，合成基因比使用克隆的自然发生基因具有许多优点。通过精确地指定核苷酸序列，可以确保表达宿主的最佳密码子使用，并根据需要加入方便的限制位点，促进盒突变和亚克隆。即使是一个相对较小的基因的合成通常也被认为是一项困难和耗时的任务，最好留给专业实验室。本文描述了一种新的省时省力的聚合酶链式反应技术，它大大简化了基因合成过程，并具有合成比现有技术大得多的基因的潜力。传统上，合成基因是通过连接较短的寡核苷酸来组装的。通常，两条DNA链都完全合成为短重叠的寡核苷酸，经过磷酸化、退火和连接产生全长产物(Edge等人，1981；Ferretti等人，1986；Bell等人，1988)。合成成本可以通过合成代表每条链的部分序列的寡核苷酸来降低，并且在连接之前使用DNA聚合酶来填充退火物中的缺口(Rink等人，1984)。在实践中，这两种方法都获得了较低的全长产物，并且在进一步操作合成的基因之前需要通过克隆进行扩增。最近，已经描述了一种通过化学合成234个碱基的寡核苷酸并使用引物来扩增化学合成的任何全长分子的聚合酶链式反应程序(Barnet和Erfle，1990)。虽然这一过程有效地增加了能够以有用的产量直接合成的序列的长度，但与中等大小的结构基因相比，长度仍然相对较短。基因也可以通过“重叠延伸拼接”的方法组装(Higuchi等人，1988)，在这种方法中，聚合酶链式反应产物被从它们的扩增引物中提纯出来，并相互延伸以产生更大的产物。该产物同时通过加入较小的侧翼引物而扩增。我们开发了一种用于基因合成的聚合酶链式反应技术，这种技术既不需要磷酸化也不需要连接，产量高，并且有可能合成比其他现有技术更大的基因。这种方法的成本相对较低，因为只有代表每条链部分序列的寡核苷酸是化学合成的，就像在DNA聚合酶填充方法中一样。寡核苷酸
In protein engineering applications involving mutagenesis and expression of proteins from recombinant DNA, synthetic genes offer many advantages over using cloned naturally occurring genes. By precisely specifying the nucleotide sequence, optimal codon usage for the expression host can be ensured and convenient restriction sites incorporated as required, facilitating cassette mutagenesis and subcloning. The synthesis of even a relatively small gene has generally been considered a difficult and time consuming task, best left to specialist laboratories. In this paper, a new cost and labour saving PCR technique is described which greatly simplifies the process of gene synthesis and has the potential for the synthesis of significantly larger genes than currently established techniques. Synthetic genes are conventionally assembled by concatenation of shorter oligonucleotides. Generally, both DNA strands are completely synthesized as short overlapping oligonucleotides which are phosphorylated, annealed and ligated to generate the full-length product (Edge et al., 1981; Ferretti et al., 1986; Bell etal., 1988). The cost of the synthesis can be reduced by synthesizing oligonucleotides representing the partial sequence of each strand, and the gaps in the annealed product'filled in'using DNA polymerase prior to ligation (Rink et al., 1984). In practice, both methods give a low yield of the full-length product and require amplification by cloning before any further manipulation of the synthesized gene.Recently, a PCR procedure has been described in which a 234 base oligonucleotide was chemically synthesized and primers used to amplify any full-length molecules that resulted from the chemical synthesis (Barnet and Erfle, 1990). While this procedure effectively increases the length of sequence that can be synthesized directly at a useful yield, the length is still relatively short in comparison with even a moderately sized structural gene. Genes can also be assembled by the method of'splicing by overlap extension'(Higuchi etal., 1988), in which PCR products are purified away from their amplifying primers and extended against each other to produce a larger product. This product is simultaneously amplified by the inclusion of smaller flanking primers. We have developed a PCR technique for gene synthesis which requires neither phosphorylation nor ligation, gives high yields and has the potential for the total synthesis of much larger genes than other established techniques. The cost of this method is relatively low because only oligonucleotides representing the partial sequence of each strand are chemically synthesized, as in the'DNA polymerase filling-in'method. The oligonucleotides