Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries.

ABSTRACT:

Background

Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC-ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC-ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, ch

SUBMITTER: Omelina ES 

PROVIDER: S-EPMC6620194 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature