Primerize: automated primer assembly for transcribing non-coding RNA domains

Abstract

Customized RNA synthesis is in demand for biological and biotechnological research. While chemical synthesis and gel or chromatographic purification of RNA is costly and difficult for sequences longer than tens of nucleotides, a pipeline of primer assembly of DNA templates, in vitro transcription by T7 RNA polymerase and kit-based purification provides a cost-effective and fast alternative for preparing RNA molecules. Nevertheless, designing template primers that optimize cost and avoid mispriming during polymerase chain reaction currently requires expert inspection, downloading specialized software or both. Online servers are currently not available or maintained for the task. We report here a server named Primerize that makes available an efficient algorithm for primer design developed and experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides. Free access: http://primerize.stanford.edu.

Figure 1.

Schematic and runtime of the Primerize algorithm. (A–C). Schematic of the Primerize algorithm. T_m (STEP 1) and misprime matrices (STEP 2) are pre-calculated for the dynamic programming assembly. Primerize optimizes the score based on misprime and returns the best solution among a range of number of primers (STEP 3). (D). Runtime of Primerize on DNA sequences of length between 100 and 600 nucleotides. Each data point is an average of five recorded runtimes of the same sequence. Error bars are standard deviation. A quadratic fit of run time to length of sequence is shown (coefficient of determination R² is 0.9850).

Figure 2.

Input interface of the Primerize server. Primerize takes a sense-strand DNA template sequence as input. Advanced options, including minimum T_m, maximum and minimum lengths of primers, number of primers, and T7 promoter checking are available for customization.

Figure 3.

Output interface and plain text of the Primerize server. (A). Results of Primerize, including potential mispriming warnings, T7 promoter checking, run time, table of all primers and assembly scheme are returned. All results are assigned with a unique JOB_ID and are available for download as plain text. (B). Primer information written in a format that can be copied and pasted to IDT Bulk Ordering page.