A method for efficient quantitative analysis of genomic subtelomere Y' element abundance in yeasts.

Abstract

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^[-((CTmutant Y' - CTmutant control ) - (CTWT Y' - CTWT 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.