Short Technical Reports
Short Technical Reports 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, and Cheng-Hong Yang5 1Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Taiwan, 2Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 4Department of Chemical Engineering, I-Shou University, Taiwan, and 5Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Taiwan BioTechniques 46:421-431 (May 2009) doi 10.2144/000113092 Keywords: primer design; SNP genotyping; RFLP; mutagenic primer; PCR-CTPP Supplementary material for this article is available at www.BioTechniques.com/article/113092.
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.
Introduction
To date, several single nucleotide polymorphism (SNP) genotyping approaches have been reviewed (1), including polymerase Vol. 46 | No. 6 | 2009
chain reaction–restriction fragment of length polymorphism (PCR-RFLP) analysis, DNA sequencing, Taqman probes, and kinetic PCR. Other, more recently developed approaches include
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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 (8–11). 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 PCRCTPP 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), primerdimer 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 costeffective 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 http://bio.kuas.edu.tw/ prim-snping.
Materials and methods
Implementation Prim-SNPing, a web-based interface, was designed and implemented under the www.BioTechniques.com
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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 (http:// rebase.neb.com) (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. 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. Query module When a user inputs the NCBI SNP ID rs#, the sequence information for the targeted SNP is retrieved from the local NCBI dbSNP database. The contig position is retrieved from NCBI’s Reference Sequence (RefSeq) (20) to increase the length of short flanking sequences, which are usually provided in NCBI dbSNP. The Prim-SNPing system can provide a maximum flanking sequence length of 1000 bp for each SNP. This service for retrieval of flanking sequence improves the system’s flexibility for primer design.
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.
Parameter Setting
Secondary structure
Input
•
Sequence- text
•
Sequence-FASTA
•
•
•
•
File upload
•
•
•
•
Hairpin, Dimer, GC clamp
•
•
•
•
•
•
%CG, Tm
•
•
•
•
•
•
Na, Mg & Salt Con.
•
•
•
Primer length PCR product size
• •
• •
• •
•
•
Extension of SNP flanking sequences
•
•
• • •
• •
•
•
BLAST for primers
• •
•
•
•
Ratio for RE-digested RFLP length
SNP genotyping
Regular primers Output
PrimSNPing
•
SNPbox
•
PDA
Primer Z
SDP rs#ID
WASP
Primer3Plus
Table 1. Comparison of the Features of Primer Software Tools
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• •
•
•
•
•
•
RFLP-natural primers
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RFLP-mutagenic primers
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CTPP primers
•
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Primer design module The primer design module provides primers based on the settings of the input module (Figure 1). Default parameters such as primer length (∼18–26 bp), primer length difference (5 bp), GC proportion (∼40–60%), Tm calculation, hairpin, and GC clamp of primers are similar to the ones used in the literature (21–25). In general PD, primers are designed only for regular PCR without SNP genotyping consideration. For natural PD, mutagenic PD, and CTPP PD, the primer information for SNP genotyping is provided. Natural primers are designed for targeted SNPs with available RFLP enzymes. In the case of a SNP without RFLP enzyme, the mutagenic primers are designed by changing the nucleotide beside the SNP in order to determine its RFLP availability. Once the availability is confirmed, the opposite primer is designed according to a user-defined length range. In CTPP PD, the confronting two-pair primers are designed to create different lengths of the PCR product corresponding to its SNP genotype. The principle of PCR-CTPP (9) and the locations of four essential primers are illustrated in Figure 2. Annealing check module The quality of the primer set is determined by (i) GC clamp, (ii) dimer check, (iii) hairpin check, and (iv) BLAST specificity www.BioTechniques.com
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adjusted. In Figure 3G, the range for the mutagenic base of the SNP is set to ±5 bp for mutagenic PD. The annealing check module shown in Figure 3H provides highly specific and optimal primer criteria for improving the quality of the designed primers.
Figure 2. Introduction of the PCR-CTPP method (modified from References 9 and 10). In the example, the PCR-CTPP is designed to confront two-pair primers like F1, F2, R1, and R2, where F2 and R1 are opposite to the alternative nucleotide of the same SNP in both sense and antisense strands, respectively. If the SNP is A/G in the sense strand and the primer F2 only covers SNP-A (sense strand), the nucleotide in primer R1 is SNP-C (antisense strand) for its corresponding SNP-G in the sense strand. (B) Primers F1/R2 are used for PCR as a positive control designed for both alternative nucleotides of the SNP (i.e., SNP-A and SNP-G). Primers F1/R1 and F2/R2 are preferentially used for SNP-A and SNP-G, respectively. When electrophoresis is performed after the PCR, the AA type should be positive for PCR using F1/R1 and F2/R1 primers and the GG type should be positive for PCR using F2/R2 and F2/R1 primers. For heterozygous type AG, PCR using F1/R1, F2/R2, and F2/ R1 primers should all be positive. Prim-SNPing provides primers to create different lengths for these three PCR reactions. Accordingly, the SNP genotype data obtained by PCR-CTPP primers contains additional information.
for the secondary structure and specificity of primers (Figure 1). These tests ensure quality control of the designed primers. RFLP analysis module The RFLP analysis module is constructed by REBASE as described (17). Natural and mutagenic primers are processed, and the RFLP analysis module is used to verify the availability of restriction enzymes in the SNP sequence. Output module Finally, information from the primer design modules, the RFLP analysis module, and the annealing check module is integrated in the output module and made available online and via email.
Results and discussion
Input data Input data for Prim-SNPing is line-fed through the web interface for the human, rat or mouse SNP genotyping assay. The input mode contains sequence and file inputs like the ones shown in Figure 3A. When clicking on the input button, the sequence data with the SNP in [dNTP1/ dNTP2] or IUPAC format is fed into the sequence window for primer design (Figure 3B). NCBI SNP ID rs# input is also acceptable (Figure 3C) and the Vol. 46 | No. 6 | 2009
flanking sequence is adjustable in a range of up to 1000 bp. The SNP flanking sequences provided by Prim-SNPing are pre-computed; they are longer than the SNP FASTA sequences provided by NCBI dbSNP. This increased length is beneficial for the primer design. For example, the length of SNP ID rs4930098 (http://www.ncbi.nlm.nih. gov/SNP/snp_ref.cgi?rs=4930098) is 601 bp. When updating this SNP ID, Prim-SNPing provides a maximum of 1000 bp. The output results are reported online and/or via email (Figure 3D). Before the primer design output, Prim-SNPing provides further analysis using the advanced options listed in Figure 3, E–G and an annealing check as shown in Figure 3H. Figure 3E shows the common part for all primer designs provided by Prim-SNPing. In principle, PCR-RFLP for SNP genotyping depends on the restriction enzyme availability occurring in only one of the alternative nucleotides of the targeted SNP (17). Therefore, the lengths of the undigested and digested PCR products vary depending on the location of the primers. Prim-SNPing provides a novel function for the user-defined RFLP pattern for natural PD (Figure 3F). The “Ratio for RE (restriction enzyme)–digested RFLP length” is set to 3:1 by default but can be
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Output data By clicking the corresponding box in Figure 4A, the designed primers can be sorted by Tm difference and processed by the BLAST function. The user-defined primer conditions are shown in Figure 4B. Ten primers are provided for each primer design (PD) in Prim-SNPing (only one or two representative primers are shown in Figure 4). In Figures 4C and 4D, both the commercial and non-commercial restriction enzymes for SNPs in the sense (input) strand are mined from the local REBASE database (18) incorporated in Prim-SNPing. The BLAST function of a primer is accessible by clicking on it individually. The BLAST time for a primer is rather long due to the short primer length. In Figures 4C, 4D, and 4E, the Tm difference for the primer designed in Prim-SNPing is very small, ensuring a highly effective PCR reaction. The primer sequence, product size, GC number, GC percentage, Tm, Tm difference, and primer length are provided. In Figure 4D (mutagenic PD), the position of the changed nucleotide (blue color) is adjustable. Figure 2 serves as a reference to determine the relative location of the primers designed in CTPP PD (Figure 4E). In brief, the primers F2 and R1 as described (9) represent the default SNP-containing primers that confront each other for the alternative nucleotide of the SNP. The Prim-SNPing system narrows the Tm difference down to within 1°C via computation, a value in accordance with requirements listed in the PCR-CTPP literature (9). Finally, the system provides the BLAST check for the sequences of primers rather than BLAST check for the sequence of PCR amplicon (Figure 4F). comparison of some primer design software tools with the Prim-SNPing Many primer design tools have been developed. Primer 3 (25) is one of the earliest and most commonly used primer design software tools. Primer Design Assistant (PDA) (26) is a web interface primer design service, which incorporates thermodynamic theory to evaluate the fitness of primers. Recently, Primer3Plus (27), an enhanced web interface of Primer 3, was introduced. WASP (28) www.BioTechniques.com
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Figure 3. Input data for Prim-SNPing. (A) Input mode. Sequence and file input are acceptable for all primer tools in Prim-SNPing. (B) IUPAC format or [dNTP1/dNTP2] format (e.g., [A/C] or M) for SNPs within the input sequence are acceptable. When inputting data, typing empty space does not interfere with the design. SNPs for human, mouse and rat genomes are included. (C) SNP ID input for primers used in SNP genotyping. The SNP FASTA sequence for the SNP flanking region is easily uploaded by clicking the “enter” box after keying in the SNP rs# ID of NCBI dbSNP. The SNP flanking length is adjustable in the range of 500–1000 bp. (D) A report of the results is provided online and by email. (E) Common constraints for primer design. Several common criteria for primer design in general PD, natural PD, mutagenic PD and CTPP PD are included. The system uses default and user-defined settings or mixed selections of both. (F) Special input constraint of natural PD. The length of RFLP fragments is user-adjustable. Information similar to gel analysis is provided. (G) Special input constraint of mutagenic PD. The position of the artificially changed nucleotide in the mutagenic primer is selectable. (H) Annealing check for all PD tools in Prim-SNPing.
is a web-based allele-specific PCR assay designing tool for detecting SNPs and mutations. SNPbox (29) is a modular software package for large-scale primer design. Primer Z (30) is a streamlined
primer design tool for promoters, exons, and human SNPs. However, none of these tools contains a SNP genotyping service, such as restriction enzyme mining for RFLP genotyping. With the exception
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of Primer Z, none of these tools provides a BLAST function. Therefore, these tools cannot check for possible multiple annealing in the genome to prevent primers from nonspecific binding to genomic DNA. The features of the primer design software tools are compared, as shown in Table 1. SNPicker (31) is another primer design tool for SNP genotyping, which uses REBASE (18) to automatically scan for all possible designs of mutagenic primers. Unfortunately, it is not web-based. FESD (32), on the other hand, identifies flanking sequences for SNP IDs, but a primer design function is not included. Given the lack of functions in these individual software applications, it is evident that there is a need for the development of an entirely online-based and integrated software package for SNP genotyping with primer design functions. In this study, we focus on the software development of cost-effective SNP genotyping, for which input and output are entirely online-based. The advanced options (Figure 3E) and annealing check function (Figure 3H) vastly enhance the quality of the designed primers. Acceptable sequence formats for Prim-SNPing are copy-andpaste, file upload in a freely selectable format, and SNP input as [dNTP1/ dNTP2] or IUPAC code. Prim-SNPing also provides SNP ID rs# input in NCBI dbSNP (19) for natural PD, mutagenic PD, and CTPP PD. In SNP Cutter (16), a special format is required for the SNP input sequence. SNP Cutter provides online input, but results output is only available via email. The performance of Prim-SNPing–designed primers for the general PD, natural PD, mutagenic PD, and CTPP PD were all successfully tested for at least two different PCR amplicons from different genes for each PD (data not shown). As an innovation, the length pattern of the RFLP using natural PD is user-adjustable (Figure 3F), and the provided RFLP enzymes for a target SNP are confirmed to be absent within the generated PCR product. These features play an important role in SNP genotyping. If the system-provided RFLP enzymes occur within the PCR product using designed primers, then the digested patterns are changed. In other words, the PCR products are cut at more than only one site by the restriction enzyme, resulting in an undesirable complexity of the RFLP genotyping process. Some characteristics of the PCR-CTPP method have been reported www.BioTechniques.com
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(grant nos. NSC97-2311-B-037-003MY3, 97-2622-E-151-008-CC2, NSC96-2221-E-214-050-MY3, NSC962311-B037-002, NSC96-2622-E214004-CC3, 96-2622-E-151-019-CC3, and KMU-EM-97-2.1a). The authors declare no competing interests.
References
Figure 4. Output data of Prim-SNPing. (A) List order and BLAST choice. The list order of primer appears randomly. Primers sorted by Tm difference (Tm-diff) are acceptable. BLAST function of primer is performed by clicking the respective button. (B) User-defined primer condition. The conditions are the same as those for the user input in the primer design constraint under advanced options shown in Figure 3E. (C) Results of natural primer design (PD). The system shows only commercial and non-commercial restriction enzymes for the sense (input) strand only by default. Natural PD is similar to general PD, but general PD (not shown) is not capable of RFLP. (D) Results of mutagenic PD. The blue letter indicates the artificially changed nucleotide, and the nucleotide of the IUPAC code indicates the SNP of interest (marked by red color). (E) Results of CTPP PD. The red-colored letters for nucleotides C and A at the 3′ ends of forward 1 (F1) and reverse 2 (R2) primers indicates the alternative nucleotide of the same SNP in opposing orientation. (F) BLAST check for primer sequences. The system-designed primers can be selected individually to perform the BLAST function.
(9). We suggest that the melting temperature (Tm) should be the same for the four primers. However, PCR is often conducted using two or three different annealing temperatures to determine ideal conditions and it is difficult to predict which combination is superior for genotyping. The process is very similar to designing ordinary PCR workflows. Accordingly, the PCR protocol requires gradient PCR to be performed in order to determine optimal conditions. Specificity is also of importance when setting PCR-CTPP conditions (9). Often, a weak band for the nonexistent allele is observed. As long as the contrast of the bands between the existing and nonexistent alleles is high Vol. 46 | No. 6 | 2009
enough, correct genotyping is not easy. Accordingly, the CTPP method eliminates the need for a separate digestion step in RFLP, and is therefore faster and cheaper to perform. Prim-SNPing also provides a BLAST function for primer sequences, so the specificity of designed primers is controlled and improved. An added benefit of Prim-SNPing is the integrated primer design function for cost-effective SNP genotyping, including PCR-RFLP and PCR-CTPP.
Acknowledgements
This work was supported in part by the National Science Council in Taiwan
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