All instances of TAG stop codons are re-coded to TAA to ‘free up’ a codon in the final strain for the introduction of a non-genetically encoded amino acid.
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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.
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We recommend downloading the newest version of Flash here, but we support all versions 10 and above. Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint.
Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.
To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call q PCRTag 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.