Bridge-overlap-extension PCR method for constructing chimeric genes.
Bridge-overlap-extension PCR method for constructing chimeric genes.
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DOI：
10.2144/99266bm17
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发表时间：
1999-06
期刊：
影响因子：
2.7
通讯作者：
R. Mehta;J. Singh
中科院分区：
文献类型：
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作者：
R. Mehta;J. Singh
我们知道，这种桥-重叠-延伸PCR方法可以有几种应用，如插入表位标签、核定位信号、分泌或蛋白水解信号、插入DNA结合区或其它功能元件到目的蛋白中。该方法的一个显著特征是，通过使用重叠寡核苷酸并产生桥接步骤，我们在不存在任何野生型模板的情况下合成了含有与杂交PCR产物重叠的较长引物。这有助于避免使用模板DNA，并因此避免在PCR步骤期间寡核苷酸与野生型模板重新退火的问题。获得正确克隆的100%效率表明该技术的成功。此外，当将该杂合基因构建体克隆到表达载体中并转化到合适的粟酒裂殖酵母菌株中时，观察到前体（P-因子和SK的融合体）和成熟SK的高水平表达。因此，该新方法可以作为构建嵌合基因的替代方法。
We understand that this bridge-overlap-extension PCR approach can have several applications, such as insertion of epitope tags, nuclear localization signals, secretory or proteolytic signals, insertion of DNA-binding regions or other functional elements into proteins of interest.The sequence element to be inserted can be of small size or long both being within the limits of efficient oligonucleotide synthesis. A distinct feature of this method is that by using overlapping oligonucleotides and bringing about a bridging step, we carry out the synthesis of a longer primer that contains an overlap with the hybrid PCR product, in the absence of any wild-type template. This helps in avoiding the use of template DNA and, therefore, the problem of re-annealing by oligonucleotides to wild-type template during the PCR steps altogether. The success of the technique is indicated by the 100% efficiency of obtaining the correct clones. Moreover, a high level of expression of precursor (fusion of P-factor and SK) and mature SK was observed when the hybrid gene construct, was cloned into an expression vector and transformed in suitable strains of S-pombe- Therefore, this new approach could serve as an alternative method for construction of chimeric genes.