methBLAST and methPrimerDB: web-tools for PCR based methylation analysis
© Pattyn et al; licensee BioMed Central Ltd. 2006
Received: 21 March 2006
Accepted: 09 November 2006
Published: 09 November 2006
DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.
To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast.
We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.
Alterations in the patterns of DNA methylation are among the earliest and most common events in tumorigenesis [1, 2]. In the mammalian genome, methylation takes place mostly at cytosine bases that are located 5' to a guanosine in a CpG dinucleotide. While this dinucleotide is generally underrepresented in the genome, short regions are found that are rich in CpG content. Such CpG-rich regions are part of gene promoters and are coined CpG islands . Both global hypomethylation and regional promoter hypermethylation have been described in a wide spectrum of cancers . Hypomethylation (or absence of methylation) of CpG islands increases potential gene activity, whereas hypermethylation of these promoter-containing CpG islands is associated with decreased gene activity or silencing . The development of efficient and accurate methods to study cytosine methylation is therefore of critical importance in understanding the role of DNA methylation in the development and progression of cancer. Furthermore, methylation markers open perspectives for earlier detection of malignancies and possible better prognostic assessment of the patients .
Several methods have been described for evaluation of cytosine methylation including digestion of DNA with methylation-sensitive restriction enzymes followed by Southern blotting or polymerase chain reaction (PCR) . Southern blotting requires large amounts of high molecular weight DNA, which limits the use of this technique. The above mentioned limitations are counteracted by performing PCR, but still both methods rely on a complete enzymatic digestion of the DNA in order to prevent false-positive results. Instead of using methylation-sensitive restriction enzymes, other methods are based on sodium bisulphite treatment of the DNA to introduce methylation-dependent sequence differences into the genomic DNA. Sodium bisulphite converts unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Nowadays, the most frequently used DNA methylation analysis methods employ a combination of bisulphite treatment and PCR. The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) method incorporates amplification of bisulphite-treated DNA, followed by a quantification of the ratio of methylated versus unmethylated cytosines at CpG sites . An alternative method, called combined bisulphite restriction analysis (COBRA), uses standard sodium bisulphite PCR treatment followed by restriction digestion and a quantitation step . A more widespread procedure combines a bisulphite treatment and PCR-single-strand conformation polymorphism analysis (Bisulphite-PCR-SSCP or BiPS) . In a first step, the converted DNA is amplified with primers that have no CpG sites in the corresponding region of the original DNA, and as such amplify both unmethylated and methylated DNA. Sequence differences between amplified products from unmethylated and methylated DNA are visualised on a SSCP gel. The fourth and one of the most popular methods is methylation-specific PCR (MSP) . It heralded a breakthrough in speed and sensitivity for gene methylation analysis. After bisulphite conversion, PCR is performed using primers that distinguish methylated from unmethylated DNA. Unlike the procedures using restriction enzymes, MSP can be used to analyse any specific CpG site by appropriate primer design and it is not prone to false-positive results. MSP is very sensitive, permitting the analysis of small and heterogeneous samples, including paraffin-embedded material. A fifth method applies the use of a sequencing strategy to analyse the methylation status a target sequence (bisulphite sequencing or BiSeq) . Bisulphite converted DNA is amplified by PCR and subsequently sequenced to assess the methylation status of individual CpG's by sequence comparison with a reference sequence. A cloning step is introduced before the sequencing if the starting material contains a mixture of cells with different methylation levels. Although the above described PCR-based DNA methylation analysis methods are easy to use, sensitive and specific, the design and experimental validation/optimisation of the primers is often difficult, labour intensive, and excludes a certain level of standardization and uniformity. To reduce the number of difficult or even unsuccessful experimental PCR optimizations, we developed methBLAST to quickly assess the specificity of a primer pair prior to the experimental evaluation step, very much like the widely accepted (or even obligated) conventional PCR primer specificity analysis using default BLAST. Another important problem encountered during methylation analysis is the difficulty to retrieve methylation assay information for a given gene of interest by normal literature search tools. Therefore, we developed a public repository holding essential assay information (including primer sequences) for the four major PCR-based methods for DNA methylation analysis of human, mouse and rat genomes.
Results and discussion
methBLAST results of a panel of assays from methPrimerDB (1–12) and literature (13–14).
Best hit when both primers align on correct target
Sequence comparison of primers from two CDKN2A MSP assays
Ref.| methPrimerDB ID
Forward primer sequence
Reverse primer sequence
 | methPrimerDB ID 17
 | methPrimerDB ID 17
 | -
 | -
GACCCCC GAACCGCGACCC TAA
If a custom designed PCR methylation assay passes the in silico specificity requirements (determined by methBLAST) and further experimental evaluation, submission of the assay information in methPimerDB is encouraged. In addition, authors of publications in which methylation-specific PCR, Bisulphite-PCR-SSCP, Ms-SNuPE, COBRA or BiSeq assays are developed, are kindly invited to submit their validated primer sequences. On-line data submissions are possible after free registration. During registration, personal submitter details are provided, after which an email is sent with the login name and a temporary password. By changing this password to a more convenient one, the registration is complete and the user can log in to the system and submit primer sets. For submission of large datasets, a compressed file is available in the download section of the website which contains the guidelines to complete an empty provided table with the required information.
We are planning to implement an additional feature in methPrimerDB to store the valuable feedback on assay performance from users who tested an assay from the database. The extension of the submitter's feedback section with the experimental evaluation details provided by the submitter as well as user's feedback will allow a better assessment of the quality of an individual assay. Although methPrimerDB is developed to let authors submit their own validated assays, we will populate the database in the near future with manually reviewed assays from recent literature.
methBLAST and methPrimerDB are web-tools to improve the design and use of PCR-based methylation assays. A sequence homology search for methylation primers with methBLAST enables specificity assessment before experimental evaluation of a new assay. To reduce the labour-intensive design of new assays, validated methylation assays can now be stored and retrieved in methPrimerDB, a public accessible database. The database is intended to be a search portal for validated methylation assays and aims to establish a certain level of standardization and uniformity in the use of PCR based methylation assays.
Both systems run on an Apache web server in a Linux environment. methBLAST is based on NCBI's BLAST server. The databases are generated by an in house developed Perl script (available upon request) converting a subset of the NCBI's nt database that contains all non-redundant GenBank+EMBL+DDBJ+PDB nucleotide sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences). methPrimerDB data is stored and managed by an Oracle 9i relational database management system. The web interface to query the database is based on PHP scripts using the Oracle Call Interface (OCI). The database information and passwords are protected by the Oracle database management system which controls the access rights to the different tables.
Availability and requirements
We greatly acknowledge the help and insightful suggestions from Christoph Grunau and Stephen Altschul in the development and evaluation of methBLAST. The authors are most grateful for support from BioScope-IT, a Bioinformatics Service Project within the context of the Flemish Innovation Network funded by the 'Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen' (IWT 040571 BIO-IT service project). Filip Pattyn is a Research Assistant and Jo Vandesompele a Postdoctoral Researcher of the Research Foundation – Flanders (FWO – Vlaanderen). Jasmien Hoebeeck is supported by the Vlaamse Liga tegen Kanker through a grant of the Stichting Emmanuel van der Schueren. This study is supported by GOA-grant 12051203, FWO-grant G.0185.04, G.1.5.243.05 and G.0106.05, and a research grant from the Childhood Cancer Fund 'Kinderkankerfonds' (a non-profit childhood cancer foundation under Belgian law). The Belgian EMBnet Node is funded by the Belgian Science Policy. This text presents research results of the Belgian program of Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming (IUAP).
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