SNP-RFLPing: restriction enzyme mining for SNPs in genomes
- Hsueh-Wei Chang†1,
- Cheng-Hong Yang†2,
- Phei-Lang Chang3,
- Yu-Huei Cheng2 and
- Li-Yeh Chuang4Email author
© Chang et al; licensee BioMed Central Ltd. 2006
Received: 04 September 2005
Accepted: 17 February 2006
Published: 17 February 2006
The restriction fragment length polymorphism (RFLP) is a common laboratory method for the genotyping of single nucleotide polymorphisms (SNPs). Here, we describe a web-based software, named SNP-RFLPing, which provides the restriction enzyme for RFLP assays on a batch of SNPs and genes from the human, rat, and mouse genomes.
Three user-friendly inputs are included: 1) NCBI dbSNP "rs" or "ss" IDs; 2) NCBI Entrez gene ID and HUGO gene name; 3) any formats of SNP-in-sequence, are allowed to perform the SNP-RFLPing assay. These inputs are auto-programmed to SNP-containing sequences and their complementary sequences for the selection of restriction enzymes. All SNPs with available RFLP restriction enzymes of each input genes are provided even if many SNPs exist. The SNP-RFLPing analysis provides the SNP contig position, heterozygosity, function, protein residue, and amino acid position for cSNPs, as well as commercial and non-commercial restriction enzymes.
This web-based software solves the input format problems in similar softwares and greatly simplifies the procedure for providing the RFLP enzyme. Mixed free forms of input data are friendly to users who perform the SNP-RFLPing assay. SNP-RFLPing offers a time-saving application for association studies in personalized medicine and is freely available at http://bio.kuas.edu.tw/snp-rflp/.
SNP genotyping is essential for association studies in personalized medicine. Although many high-throughput SNP genotyping methods have been reported, lots of researchers still report their SNP genotyping by restriction fragment length polymorphism (RFLP). NEBcutter  can provide the RFLP information for any input sequences using REBASE information . However, it is not convenient for SNP related sequences. To discriminate one SNP in a RFLP assay, the restriction enzymes have to recognize only one of the SNP containing sequences. Therefore, the users have to input data twice for each SNP related sequence when checking for the available restriction enzymes. On the dbSNP of NCBI , each SNP is named in reference cluster IDs (rs) and in NCBI assay IDs (ss). Users can input the SNP ID, gene name (HUGO) or gene ID for Entrez gene in NCBI to get the SNP with its flanking sequences using NEBcutter . However, it is time consuming if a gene like TP53 contains hundreds of SNPs.
In this paper, we present the web-based integrated system called SNP-RFLPing for SNP ID information and its availability for restriction enzymes. Users can input any formats of SNPs including NCBI dbSNP rs or ss ID, HUGO gene name and gene ID for Entrez gene in NCBI . Then, the availability of restriction enzymes as well as SNP-related information can be presented. It also functions for user-defined SNPs, which are not reported in the NCBI database. For large data of SNP IDs or gene IDs, SNP-RFLPing provides a file upload service to perform the RFLP assay for efficient screening of SNP-RFLP enzymes in association studies.
SNP related information is provided for the RFLP assay including the SNP ID, species, contig-position, heterozygosity, function, protein residue (P), codon position (C), and amino acid position (A) (Figure 3A). It may be helpful for the users to select interesting SNP targets for association studies. The analyzed SNP can be selected as a whole or partially at the square box. Then, the RFLP availability of the restriction enzymes for the input SNP-containing sequence (marked as +) and its complementary sequence (marked as -) is shown separately in Figure 3B. The commercial and non-commercial restriction enzymes shown in Figure 3C are linked to restriction enzyme databases REBASE . SNP-RFLPing provides a mutagenic (or mismatched) primer for a SNP in which a suitable restriction enzyme can not be found naturally. The optimal primer design follows criteria as described [7, 8], such as melting temperature, length, and base composition. The primer opposing to the mutagenic primer and the natural primer sets can be designed using Primer3 , which is hyperlinked in the software.
Comprehensive table for comparison of the features of RFLP related software.
Type of program
Flanking sequence length
Design of mutagenic primers
1.rs# IDs, ss# IDs.
2.Many IDs per line (separated by comma symbol).
3.HUGO gene name.
4.Entrez Gene ID.
5.SNP-in-sequences of IUPAC or [dNTP/dNTP] format.
6.Multiple mixed forms: rs#, ss# and sequences are accepted (separated by comma or enter key).
1.On-line and/or email.
2.SNP information provided, e.g., contig position, heterozygosity, function, protein codon.
3.RFLP information, e.g., recognition site, cutting position and strand of restriction enzyme. (visualization of the result).
4.Gene-SNP-RFLP function. (Users can input the gene name and the system can provide all RFLP SNPs of the gene)
1.RFLP information, e.g., recognition site, cutting position and strand of restriction enzyme.
2.no SNP information.
2.no SNP related input.
2.no SNP information.
SRP Opt 
2.microbial genome only.
1.not for SNP genotyping.
2.selection of forensic markers.
PIRA-PCR Designer 
2.no SNP information.
SNP cutter 
2.one ID per line.
3.Specific sequence format (additional software needed).
2.no SNP information.
1.upload input only.
5.no sequence input.
2.no RFLP information.
2.FASTA format only.
1.no auto-mining for RFLP enzyme.
In silico software http://www.in-silico.com/restriction/ 
2.sequences with SNP or mutation.
3.unaligned or multiple prealigned sequences are accepted.
2.SNP position in sequence.
3.RFLP information (including cutting position, selectable function for minimum recognition size, type of enzymes and commercial sources.).
4.multiple SNPs can be compared simultaneously.
5.other useful related tools provided.
In the SNP-RFLPing server, more input items are provided, including: rs#, ss#, gene name, and ID for human, mouse and rat genomes. It is very convenient for a user to check the available restriction enzyme for each gene of interest, both online and per email. To our knowledge, SNP-RFLPing is the first software to link the gene name and its SNP-RFLP restriction enzyme. It's not necessary to search all SNPs of a certain gene from the NCBI dbSNP  before putting all these SNPs into a suitable SNP-RFLP software, like SNP cutter . SNP500Cancer  also provides SNP searching by genes, but doesn't provide the RFLP function, and the coverage of SNPs is limited to human cancer-related genes. In SNP-RFLPing, only one step is needed without transforming specific formats before assay. This design will speed up the screening with SNP-RFLPing compared to other available software.
In addition to RFLP enzymes, RFLP genotyping also needs the primers for PCR-RFLP. Softwares like PIRA-PCR , SNP cutter , and software from the in-silico company  can provide a design function for mutagenic primers (Table 1). Similarly, SNP-RFLPing provides the newly developed mutagenic primer designer. We also provide a hyperlink to the freely available software Primer3  for the design of primers opposing to mutagenic primer and the natural primer sets. The path for primer design in SNP-RFLPing will be integrated in the future. Alternatively, we recommend a user to use SNP-RFLPing software coupled with other commercial primer designers, e.g., Beacon Designer 4 (Premier Biosoft International, CA), which are usually unable to provide the RFLP information, but provide a fast and friendly natural primer design for each SNP.
The web-based software, SNP-RFLPing, can solve the input format problems inherent in similar software, and greatly simplify the procedure for providing the RFLP enzyme. A novel function of SNP-RFLPing is that it can accept any common input formats to check the RFLP availability in human, mouse, and rat genomes. In addition, the searching of SNP and RFLP information by gene name is a very powerful tool for association studies with a target gene. In conclusion, it is time-saving and user-friendly to use SNP-RFLPing for association studies in personalized medicine.
Availability and requirements
Project name: SNP-RFLPing: restriction enzyme mining in genomes.
Project home page: http://bio.kuas.edu.tw/snp-rflp/
Operating system(s): Microsoft Windows XP
Programming language: Java
Other requirements: Java 1.5.0, Tomcat 5.5, SQL server 2000, MySQL 4.0
License: none for academic users.
For any restrictions regarding the use by non-academics please contact the corresponding author.
List of abbreviations
single nucleotide polymorphism
restriction fragment length polymorphism
National Center for Biotechnology Information
Human Genome Organization
The Restriction Enzyme Database
This work is partly supported by the National Science Council in Taiwan under grant NSC94-2622-E-151-025-CC3, NSC93-2213-E-214-037, NSC92-2213-E-214-036, NSC92-2320-B-242-004, NSC92-2320-B-242-013 and by CGMH fund CMRPG1006.
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