多片段拼接法克隆人表皮生长因子受体2基因
Cloning of Human Epidermal Growth Factor Receptor 2 Gene by Multi Fragment Assembly

鉴于从cDNA库中克隆长基因较困难的现实，采取分步扩增与DNA组装相结合的方式，提出一种长基因克隆解决方案。以克隆人表皮生长因子受体2基因为例，先分别扩增出该基因的三个片段F1、F2和F3。通过重叠延伸PCR融合前两个片段得到F12，再利用双片段连接法将F12与F3一起连接插入载体以构建完整基因。从双片段连接物中筛选出上百个菌落，经检测和序列测定，得到一个序列完全准确的克隆。对于难以直接克隆的长基因，可先扩增其各个片段，再将它们拼接成完整基因。其中双片段连接法可突破严格的酶切位点要求对单片段顺序连接的限制，再辅以重叠延伸PCR融合法，即可实现多个基因片段的正确拼接。
It was difficult to clone long genes from cDNA library due to a series of factors, such as mRNA instability and the low efficiency of reverse transcription. Combining partial amplification with DNA assembly, an optimal solution was proposed for cloning lone gene that was difficult to amplify from cDNA directly. Taking the human epidermal growth factor receptor 2 (HER2) gene as an example, three fragments of HER2 were amplified firstly. F1 and F2 were fused into F12 by overlap extension PCR, and then enzyme cleaved F12 and F3 were linked together into vector, so as to recombine the whole HER2 gene. Hundreds of colonies were screened out from double fragments ligation and one positive clone matching completely to HER2 sequence was detected. For long gene that was difficult to clone directly, it was feasible to amplify its segments and then assemble them into a complete one. Double fragments ligation breaking the rigorous demand of restriction sites of step by step ligation, in the assistance of overlap extension PCR, could join DNA fragments into a complete gene.