qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping
qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping
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DOI：
10.3791/52941
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发表时间：
2015-05-01
影响因子：
1.2
通讯作者：
Boeke, Jef D.
中科院分区：
文献类型：
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作者：
Mitchell, Leslie A.;Phillips, Nick A.;Boeke, Jef D.
合成酵母基因组计划 (Sc2.0) 旨在构建 16 条设计酵母染色体并将它们组合成单个酵母细胞。迄今为止，已经构建了一种合成染色体synIII(1)和一种合成染色体臂synlXR(2)，并且在不存在相应野生型染色体的情况下验证了它们的体内功能。 Sc2.0 染色体的一个重要设计特征是引入了 PCRTags，它是开放阅读框 (ORF) 内的短的、重新编码的序列，能够将合成染色体与其野生型对应染色体区分开来。 PCRTag 引物选择性地与合成或野生型染色体退火，并且可以使用简单的 PCR 测定来测试每种类型 DNA 的存在/不存在。 PCRTag 测定的标准读数是通过琼脂糖凝胶电泳评估扩增子的存在/不存在。然而，平均 PCRTag 扩增子密度为每 1.5 kb 1 个，基因组大小接近 12 Mb，完整的 Sc2.0 基因组将编码大约 8,000 个 PCRTag。为了提高通量，我们开发了一种基于实时 PCR 的 PCRTag 基因分型检测方法，我们称之为 qPCRTag 分析。该工作流程在 1,536 多孔板中指定 500 nl 反应，使我们能够在单个实验中使用合成和野生型引物对测试多达 768 个 PCR 标签。
The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synlll(1), and one synthetic chromosome arm, synlXR(2), have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of similar to 12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.