Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously
Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously
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DOI：
10.1093/nar/gnh014
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Ladisch, S
中科院分区：
文献类型：
--
作者：
Shevchuk, NA;Bryksin, AV;Ladisch, S
提出了一种精确组装长达20kb的线性DNA结构的方法。这种方法，我们称之为长多次融合，已被用于同时组装多达4个片段(最终产物为10.8kb)，提供了对长PCR和重叠延伸PCR组合的额外改进。该方法是基于具有校对活性的PFU聚合酶混合物。我们成功地组装了7个不同的线性构建体(通过测序证实)，范围从3到20kb，包括两个20kb的产物(来自11、1.7和7.5kb的片段)，两个10.8kb的构建体，以及两个分别为6.1和6.2kb的构建体。聚合酶链式反应融合的准确性大于或等于每6.6kb一个错误，这与聚合酶链式反应混合的预期错误率一致。该方法有望为各种复杂的基因工程项目提供便利，这些项目需要多个片段的精确框内组装，例如在人类细胞中敲除体细胞或为疫苗研究创建完整的病毒基因组。
A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.