High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding.

ABSTRACT: Cis-regulatory elements (CREs) are one of the important factors in controlling gene expression and elucidation of their roles has been attracting great interest. We have developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our approach, mutant CREs with unique barcode sequences were obtained by biased randomization in a single PCR amplification. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within the range of 0 to 5. The DNA library and subsequent transcribed RNA library were sequenced by next generation sequencers (NGS) to quantify transcriptional activity of each mutant. We succeeded in producing a randomized T7 promoter library w

SUBMITTER: Komura R 

PROVIDER: S-EPMC5937735 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature