Moqtaderi Zarmik, Geisberg Joseph V
Harvard Medical School, Boston, Massachusetts.
Curr Protoc Mol Biol. 2013 Oct 11;104:13.10C.1-13.10C.17. doi: 10.1002/0471142727.mb1310cs104.
Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene.
传统上，在酵母基因组的目标位点引入特定新突变的方法涉及通过多个克隆步骤制备破坏或基因替换盒。用于靶向这些盒或整合载体的序列通常有几百个碱基对长。各种更新的方法依赖于设计含有较短序列尾（约50个核苷酸）的定制PCR寡核苷酸，以靶向感兴趣的位点。这些技术无需克隆步骤，并允许通过PCR扩增构建诱变盒。这种盒可用于基因缺失、表位标签或位点特异性诱变。这些策略在几个方面有所不同，最显著的是它们是否允许选择标记的重复使用以及在目标位点附近是否留下额外序列。本单元总结了不进行克隆的酿酒酵母基因座靶向诱变方法，包括基于PCR的等位基因替换、完美诱变和MIRAGE。接下来，提供了一种基于完美诱变PCR和寡核苷酸的诱变方法的方案，该方法在产生同一基因的几种不同突变等位基因方面具有特别的优势。
