Prim-SNPing: A Primer Designer for Cost-Effective SNP Genotyping

Many kinds of primer design (PD) software tools have been developed, but most of them lack a single nucleotide polymorphism (SNP) genotyping service. Here, we introduce the web-based freeware "Prim-SNPing," which, in addition to general PD, provides three kinds of primer design functions for cost-effective SNP genotyping: natural PD, mutagenic PD, and confronting two-pair primers (CTPP) PD. The natural PD and mutagenic PD provide primers and restriction enzyme mining for polymerase chain reaction–restriction fragment of length polymorphism (PCR-RFLP), while CTPP PD provides primers for restriction enzyme–free SNP genotyping. The PCR specificity and efficiency of the designed primers are improved by BLAST searching and evaluating secondary structure (such as GC clamps, dimers, and hairpins), respectively. The length pattern of PCR-RFLP using natural PD is user-adjustable, and the restriction sites of the RFLP enzymes provided by Prim-SNPing are confirmed to be absent within the generated PCR product. In CTPP PD, the need for a separate digestion step in RFLP is eliminated, thus making it faster and cheaper. The output of Prim-SNPing includes the primer list, melting temperature (Tm) value, GC percentage, and amplicon size with enzyme digestion information. The reference SNP (refSNP, or rs) clusters from the Single Nucleotide Polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI), and multiple other formats of human, mouse, and rat SNP sequences are acceptable input. In summary, Prim-SNPing provides interactive, user-friendly and cost-effective primer design for SNP genotyping. It is freely available at http://bio.kuas.edu.tw/prim-snping.