Subtelomere Y′ elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time‐consuming. By multiple sequence alignment analysis of all 19 subtelomere Y′ elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real‐time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real‐time quantitative PCR analysis, so the relative Y′ element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y′ element copy numbers across all different chromosomes using the formula: 2^[−((CT_{mutant Y′} − CT_{mutant control}) − (CT_{WT Y′} − CT_{WT control}))]. This novel quantitative subtelomere amplification assay across chromosomes by real‐time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y′ element recombination events in survivors derived from telomerase deficiency or recruitment failure.

中文翻译：

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一种有效定量分析酵母中基因组亚端粒Y'元素丰度的方法。

亚端粒Y'元件通过同源重组得以扩增，以维持出芽酵母酿酒酵母的存活和分裂。然而，当前的测量亚端粒结构的方法使用带有标记的特异性探针的Southern印迹法，这既费力又费时。通过对沉积在酵母基因组SGD数据库中的已测序S288C菌株的13条染色体上所有19个亚端粒Y'元件的多序列比对分析，我们鉴定了12个共有和相对较长的片段以及14对独特的引物，用于实时定量PCR分析。使用PAC2或ACT1这些引物位于V和VI染色体的着丝粒附近作为内部对照，被应用于实时定量PCR分析，因此针对亚端粒Y'元件拷贝计算了相对于野生型（WT）细胞标准化的相对Y'元件强度使用以下公式计算所有不同染色体的数目：2 ^ [-（（CT_突变Y' -CT_突变对照）-（CT _{WT Y'} -CT _WT对照））。与传统的Southern印迹法相比，这种新颖的实时定量PCR跨染色体定量亚端粒扩增测定方法比传统的Southern印迹法分析端粒酶缺乏或募集失败的幸存者中Y'元件重组事件更为简便和灵敏。