qPCRTag analysis - A high throughput, real time PCR assay for Sc2.0 genotyping

Leslie A. Mitchell, Nick A. Phillips, Andrea Lafont, James A. Martin, Rupal Cutting, Jef D. Boeke

    Research output: Contribution to journalArticle

    Abstract

    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, synIII1, and one synthetic chromosome arm, synIXR2, 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 ~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.

    Original languageEnglish (US)
    Article numbere52941
    JournalJournal of Visualized Experiments
    Volume2015
    Issue number99
    DOIs
    StatePublished - May 25 2015

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    Keywords

    • Cobra
    • Echo
    • Genotyping
    • Issue 99
    • LightCycler 1536
    • Molecular biology
    • PCRTag
    • Real time PCR
    • Saccharomyces cerevisiae
    • Sc2.0
    • Synthetic biology

    ASJC Scopus subject areas

    • Neuroscience(all)
    • Chemical Engineering(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Immunology and Microbiology(all)

    Cite this

    Mitchell, L. A., Phillips, N. A., Lafont, A., Martin, J. A., Cutting, R., & Boeke, J. D. (2015). qPCRTag analysis - A high throughput, real time PCR assay for Sc2.0 genotyping. Journal of Visualized Experiments, 2015(99), [e52941]. https://doi.org/10.3791/52941