Template-Switching Enhances Primer Extension Analysis of Transcription Start Sites
- Author(s): Lin, Wilson;
- Advisor(s): Kadonaga, James T;
- et al.
Proximal to transcription start sites, core promoters serve as key regulators of gene expression by sequence-based differential recruitment of transcriptional proteins. Thus, mapping transcription start sites (TSSs) can lead to identification and characterization of core promoter elements, thereby elucidating the relationship between DNA sequence and regulation of gene expression. Using reverse transcription of RNA from active promoters, primer extension can be employed as a quantitative single-step technique for mapping TSSs with high resolution. However, very small amounts of RNA may be challenging to detect with primer extension, and background signals commonly occur due to RNA secondary structures – both issues constrain the utility of primer extension. One possible avenue for addressing these limitations is template-switching: a mechanism that has improved specificity and yield for numerous RNA assays. Thus, we sought to determine whether template-switching can be used to improve primer extension-based analysis of core promoters. In this paper, we outline obstacles and optimizations for implementing template-switching into primer extension. In modifying dNTP concentrations in primer extension to better suit template-switching, we observed highest template-switching efficiency in low overall (<1mM) dNTP concentrations with a relative excess of dCTP; compared to enzymatic alternatives for template-switching, this optimization may be both novel and cost-effective. Furthermore, we found that this template-switching primer extension approach provided both greater sensitivity and dramatically lower background than the traditional primer extension technique. Therefore, we conclude that template-switching effectively enhances primer extension analysis, thus broadening the usefulness of primer extension in studying core promoters.