MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

Abstract

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR.

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments

“… -indicates not applicable. *To calculate the melting temperature, we adopted the nearest-neighbor thermodynamic model ( 22 ). **The value ranges in this column indicate the default setting, which can be freely adjusted by users.…”

“…The large-scale computation of each round of homology testing relies on distributed data processing in MapReduce. Details and examples of Rounds 3–5 are described in a previous publication ( 22 ).…”

Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation

“…And total RNA was used to synthesize the first strand cDNA using RNA to cDNA EcoDry premix Oligo-dT (Clontech). Quantitative PCRs were performed with the use of SYBR green PCR Master Mix (Toyobo) [ 59 ] and we used an ABI 7300 real time PCR system (Applied Biosystems). GAPDH was used as the internal standard for normalization.…”

MRPrimerV: a database of PCR primers for RNA virus detection

“…To obtain all feasible and valid primers for RNA virus detection, we applied multiple filtering constraints and performed large-scale rigorous similarity testing against all human gene sequences using the MRPrimer technology (13), which returns all feasible and valid primer pairs existing in RNA virus sequences. MRPrimer performs fairly complex, large-scale processing to simultaneously check filtering constraints and perform similarity testing of all possible sub-sequences in a given database, based on a distributed MapReduce framework, resulting in design of very high-quality primers.…”

“…MRPrimer performs fairly complex, large-scale processing to simultaneously check filtering constraints and perform similarity testing of all possible sub-sequences in a given database, based on a distributed MapReduce framework, resulting in design of very high-quality primers. qPCR analysis using many primer pairs, along with the corresponding sequencing and comparative analyses, revealed that primer pairs designed by MRPrimer are stable and effective in qPCR experiments (13). For qPCR experiments as well as for single or simultaneous multiple specific RNA virus detection, we applied the same filtering constraints to all primer pairs for all RNA viruses (Table 1).…”