尽管存在将合成寡核苷酸组装成基因和基因组的有效方法,这些遭受1-3随机错误/kb的DNA的存在。这里,我们引入了一种称为共识改组的新方法,并证明了该方法可显著减少合成DNA中的随机误差。在这种方法中,错误通过种群的重新杂交被揭示为错配。DNA片段化了,在与固定的错配结合蛋白(MutS)结合后去除错配片段。剩余片段的PCR组装产生了富含输入群体的共有序列的全长序列的新群体。我们表明,两次共识改组的迭代改进了合成绿色荧光蛋白(GFPuv)克隆的群体,从大约60到>90%的荧光,并将误差降低3.5至4.3倍,最终值大约为每3500bp1个误差。此外,两次共识洗牌迭代纠正了所有成员都无功能的GFPuv克隆群体,82%的克隆具有荧光。共有改组应该有助于长DNA序列的快速和准确合成。
Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1-3 random errors/kb of DNA. Here, we introduce a new method termed
consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the
consensus sequence of the input population. We show that two iterations of
consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from approximately 60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of approximately 1 error per 3500 bp. In addition, two iterations of
consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent.
Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.