HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis

Method Summary

Described here is the web-service HTP-OligoDesigner, an online batch primer design tool for sequence-independent gene cloning and site-directed mutagenesis.

The ever-growing amount of available genome sequencing data generated by recent advances in DNA sequencing techniques is providing an inexhaustible source of uncharacterized genes to be studied by structural, biochemical, and molecular biology methods and techniques (Metzker, 2010; Mardis, 2011). Laboratories worldwide are developing and implementing high-throughput cloning and expression platforms, which include target selection, construct design, and expression screenings to identify the most viable targets in terms of soluble protein expression, yield, and activity (Stevens et al., 2001; Lesley et al., 2002; Bunkoczi et al., 2007; Sauder et al., 2008; Xiao et al., 2010; Vincentelli et al., 2011). Since primer design can be a time-consuming and error-generating task when applied to hundreds of targets, the automation of the design process is extremely convenient and attractive, especially for scientists whose experience in bioinformatics programming is not enough to create command lines for such a task. In fact, several free web-based or stand-alone primer design tools with excellent functionalities are available on the web, but most of them are only capable to analyze one sequence at a time. BatchPrimer3 (You et al., 2008), on the other hand, is endowed with a batch functionality, but it is not capable to design cloning and mutagenesis primers. Aiming to fill this gap, HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive tool for batch primer design for sequence-independent gene cloning and site-directed mutagenesis. The web tool is freely available online (Camilo et al., 2015) and implemented using JavaScript and jQuery, with all major browsers supported.

HTP-OligoDesigner generates cloning primer pairs by fixing the 5′-end nucleotide position of each primer and varying the length of the primers from 5′ end to 3′ end, according to a chosen Tm calculation algorithm. The software does not check for the existence of restriction sites within the gene sequences, which would interfere in restriction enzyme/ligation methodologies. Thus, we recommend the use of sequence-independent cloning methodologies such as Gateway^® (Life Technologies), LIC (Aslanidis and de Jong, 1990), In-Fusion™ (Clontech), and PIPE (Klock and Lesley, 2009). The Tm calculations are based on the nearest-neighbor method as described by Breslauer et al. (1986). Additionally, two options of thermodynamic data are available: from Breslauer et al. (1986) and from Sugimoto et al. (1996). Input data can be a flat text or an uploaded text file, containing coding region sequences of desired target protein genes in the FASTA format. Both cloning and mutagenic primers are designed according to the optimal Tm selected by the user with a maximum Tm variation of 3°C from the optimal value and a maximum Tm difference of 3°C between sense and antisense primers. Also, in order to promote annealing and increase amplification efficiency, the program prioritizes primers ending with G or C within the threshold of 5°C from the optimal Tm value. Concentrations of primers and monovalent cations can also be defined by the user and should preferably reflect PCR conditions. A field where extra sequences at the 5′ ends of generated cloning primers can be appended is also provided. The appended sequences are not taken into account for Tm calculation.

Mutagenic primers are generated based on inverse PCR method (Rabhi et al., 2004) in which the entire plasmid is amplified by PCR, followed by blunt-end ligation. HTP-OligoDesigner is capable of designing primers for three types of mutations: point mutation, deletion, and insertion. Desired mutations must be indicated into each sequence so that they can be joined in a flat text or uploaded text file in FASTA format. To be identified by the software, the base(s) to be changed and the new base(s) must be separated by dot and enclosed in parentheses, as depicted in Table 1. HTP-OligoDesigner provides a tool to help user convert conventional amino acids mutation notation to its own.

Output data from both mutagenesis and cloning tools are given as plain text containing the GeneID (first uninterrupted characters from FASTA header), primers sequences, lengths, and Tm values. A complete tutorial for the website can be found online.

HTP-OligoDesigner has been running for over one year and used for high-throughput cloning (Camilo and Polikarpov, 2014) and laboratory-scale mutagenesis experiments.

Author Contributions

C.M.C. idealized the tool and wrote the article. G.M.A.L. developed the web service. F.V.M. proof tested the algorithm with biological assays (data not shown) and provided useful insights. R.V.C.G. and I.P. secured the funding and contributed intellectually.