![]() Users can access all stored information (primer sequences and annotations, primer validation data, as well as gene annotations) from the PrimerBank website ( ). A total of 91 502 and 98 854 new primers for humans and mice, respectively, were designed and included in the database. Current genomic annotations from the National Center for Biotechnology Information (NCBI) databases were consolidated as a consequence of the update. In this work, we describe an updated primer design process for PrimerBank that reflects the maturation of our understanding of the murine and human genomes and reflects further refinements in the design strategy for primer selection. Thus, the design of target sequence-specific fluorescent probes has not been required, leading to significant savings. All PCR primers were designed to work with sequence-independent fluorescence detection methods such as SYBR Green-based qPCR ( 15, 16). The design of PrimerBank primers was founded in turn on an algorithm for the design of oligonucleotide probes for microarrays, with additional criteria that are specific to PCR ( 14). The PrimerBank resource and its experimentally validated 26 855 primer pairs covering most known mouse genes have previously been described ( 11–13). The high annealing temperature helps reduce non-specific amplification. To allow for an efficient high-throughput qPCR process, PrimerBank primers have been designed and validated to perform at an invariant annealing temperature of 60☌. The primers can be applied either to small-scale experiments focusing on a few genes, or to high-throughput qPCR for thousands of genes. To address the primer design challenge, we developed the PrimerBank database, which contains tens of thousands of computationally designed as well as experimentally validated primers. Multiple design algorithms have been proposed ( 7–10), but few of them have been validated experimentally. Historically, a significant obstacle to genome-wide PCR had been the inability to identify robust oligonucleotide primers that could be used under identical conditions. Real-time PCR has several major advantages over DNA microarrays, the most important being a large dynamic range, high sensitivity and high specificity ( 5, 6). ![]() However, with the appropriate design strategy, qPCR can also be performed in parallel for thousands of genes in a genome-wide fashion. Often the role of qPCR is to validate expression changes of selected genes showing profiles of interest in an initial genome-wide survey ( 3, 4). Real-time PCR is routinely employed for gene expression quantification, and has been widely used for the confirmation of results from high-throughput experiments such as DNA microarray expression profile studies or transcript abundance measurements by next-generation sequencing. Both probe-(allele-)specific as well as allele-non-specific technologies have been described. The quantitative polymerase chain reaction (qPCR) technique, also known as real-time PCR, was developed in the early 1990s and subsequently improved or modified by a large number of groups ( 1, 2). The primers and all experimental validation data can be freely accessed from the PrimerBank website. Because of their broader linear dynamic range and greater sensitivity, qPCR approaches are used to reanalyze changes in expression suggested by exploratory technologies such as microarrays and RNA-Seq. PrimerBank primers work under uniform PCR conditions, and can be used for high-throughput or genome-wide qPCR. An updated algorithm based on our previous approach was used to design new primers using current genomic information available from the National Center for Biotechnology Information (NCBI). As a result of this update, PrimerBank contains 497 156 primers (an increase of 62% from the previous version) that cover 36 928 human and mouse genes, corresponding to around 94% of all known protein-coding gene sequences. Here, we describe a major update of PrimerBank that includes the design of new primers covering 17 076 and 18 086 genes for the human and mouse species, respectively. A distinguishing feature of PrimerBank is the experimental validation of primer pairs covering most known mouse genes. We have developed a resource, PrimerBank, which contains primers that can be used for PCR and qPCR under stringent and allele-invariant amplification conditions. Optimization of primer sequences for polymerase chain reaction (PCR) and quantitative PCR (qPCR) and reaction conditions remains an experimental challenge.
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