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Prim-SNPing: a primer designer for cost-effective SNP genotyping
Hsueh-Wei Chang1, 2, 3, Li-Yeh Chuang4, Yu-Huei Cheng5, Yu-Chen Hung1, Cheng-Hao Wen1, De-Leung Gu1, Cheng-Hong Yang5
1, Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Taiwan
2, Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
3, Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
4, Department of Chemical Engineering, I-Shou University, Taiwan
5, Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Taiwan
BioTechniques, Vol. 46, No. 6, May 2009, pp. 421–431
Full Text (PDF)
Supplementary Material

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


To date, several single nucleotide polymorphism (SNP) genotyping approaches have been reviewed (1), including polymerase chain reaction–restriction fragment of length polymorphism (PCR-RFLP) analysis, DNA sequencing, Taqman probes, and kinetic PCR. Other, more recently developed approaches include SNP genotyping with fluorescence polarization detection (2), the Invader assay (3), DNA microarray (4,5), and pyrosequencing (6). Currently, most of these methods are prohibitively expensive.

Among these methods, PCR-RFLP has become one of the more commonly used methods for genotyping for genetic association studies in laboratories. PCR-RFLP requires PCR amplification and restriction enzyme digestion before electrophoresis (7). However, DNA digestion requires 3–24 h, depending on the applied restriction enzyme. Recently, a novel method, PCR with confronting two-pair primers (PCR-CTPP), has been developed to perform SNP genotyping without DNA product digestion (8,9,10,11). If the digestion step can be skipped, the SNP genotyping process is greatly accelerated. However, it has been suggested that the melting temperature (Tm) range for the primer design in PCR-CTPP is limited (9). This limitation reduces the practical use of PCR-CTPP without computational help.

Although the PCR-RFLP and PCR-CTPP methods are cost-effective, the development of computational algorithms for their primer design is necessary for researchers without a background in SNP genotyping. Fortunately, many inherent primer design problems have been solved, including the mutagenic primer problem (12,13), primer-dimer and hairpin structures by AutoDimer (14), and secondary structure and optimized downstream genotyping applications by DFold (15). SNP Cutter (16) provides both natural PD and mismatch (mutagenic) PD for PCR-RFLP genotyping with online input but email output for the result. Consequently, we have enough information to develop a well-rounded primer design tool, which can be coupled with previously introduced software, SNP-RFLPing (17) for SNP genotyping.

In this study, we introduce Prim-SNPing, an improved software tool for primer design and RFLP enzyme mining for cost-effective SNP genotyping, which supports both online input and output. Free format sequence inputs and reference SNP cluster ID number (SNP ID rs#) inputs are acceptable. We also developed a novel primer design function “CTPP PD” for SNP genotyping, which omits the digestion step for restriction enzymes. Therefore, Prim-SNPing provides user-friendly and cost-effective primer design for SNP genotyping. The software is available for free at

Materials and methods


Prim-SNPing, a web-based interface, was designed and implemented under the SQL server database system. Java server pages and Java applets are used to input data and process files between the user and the application, and to parse the data, respectively. The database structure is mainly set up by REBASE v. 607 ( (18) and NCBI dbSNP Build 125 (19), and is transformed into the MySQL format and a local database copy, respectively. We plan to update these databases annually.

Program workflow

The schematic program workflow of Prim-SNPing (Figure 1) consists of six modules: (1) Input, (2) Query, (3) Primer Design, (4) Annealing Check, (5) RFLP Analysis, and (6) Output.

Figure 1.

Flowchart of web-based Prim-SNPing. Three kinds of functions are incorporated into the input module of the Prim-SNPing system: the primer design (PD) type (general PD, natural PD, mutagenic PD and CTPP PD), the sequence input type (rs#, sequence and file input), and the primer design constraints, as indicated. The query module provides the SNP FASTA sequence and flanking sequence nearby the SNP using rs# search via NCBI dbSNP (19). In a next step, the flanking sequence of the SNP is fed into the primer design module for usage with the four PDs. Subsequently, the annealing check and RFLP analysis modules provide further analysis if selected. In the RFLP module, the SNP-containing sequences are transferred to a local database downloaded from REBASE (18), and then the RFLP availability for the sense and antisense sequences is determined. Finally, the output module displays the RFLP enzyme and primer information as indicated. Tm, melting temperature; Tm-diff, Tm difference.

Input module

Four kinds of primer design types (i.e., general PD, natural PD, mutagenic PD and CTPP PD) are provided. Except for the general PD, all other forms of PD in Prim-SNPing provide the primers for SNP genotyping in a cost-effective manner. Sequence input types include SNP ID rs# feed-in, sequence copy-and-paste, and text file upload. Primer design constraints are shown in detail in Figure 1.

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