The Kunkel method is a widely used site-directed mutagenesis strategy that introduces point mutations by annealing mutation-containing oligonucleotides to single-stranded uracil-containing DNA (dU-ssDNA) templates. The method is fast and inexpensive and has been routinely employed to generate point mutations and multi-site mutations. However, its efficiency for point mutations is highly variable. In this work, codons in both DNA templates and mutagenic oligonucleotides were optimized to lower the GC percentage (GC%) of the complementary regions, and the oligonucleotide length was also extended to reduce the GC difference between upstream and downstream regions. These modifications largely increased the mutation efficiency of single-site mutagenesis. In addition, a multi-stage cooling programme was developed in the annealing step specifically for multi-site mutagenesis, which increased the simultaneous mutation efficiency. The modifications will help in generating antibody libraries by effectively randomizing multiple CDRs simultaneously.

中文翻译：

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通过密码子优化和退火温度调节提高Kunkel法诱变效率

Kunkel 方法是一种广泛使用的定点诱变策略，它通过将含有突变的寡核苷酸与含有单链尿嘧啶的 DNA (dU-ssDNA) 模板退火来引入点突变。该方法快速且廉价，已被常规用于产生点突变和多位点突变。然而，它的点突变效率是高度可变的。在这项工作中，对 DNA 模板和诱变寡核苷酸中的密码子进行了优化，以降低互补区域的 GC 百分比 (GC%)，并延长寡核苷酸长度以减少上游和下游区域之间的 GC 差异。这些修改在很大程度上提高了单点诱变的突变效率。此外，在退火步骤中开发了多阶段冷却程序，专门针对多位点诱变，提高了同时突变的效率。这些修改将通过同时有效地随机化多个 CDR 来帮助生成抗体库。