BiSearch: Primer-design and Search Tool for PCR on Bisulfite-treated Genomes

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2005 Jan 18

PMID 15653630

Citations 82

Authors

Gabor E Tusnady

Istvan Simon

Andras Varadi

Tamas Aranyi

Affiliations

Soon will be listed here.

Abstract

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

Citing Articles

Systematic review on the DNA methylation role in endometriosis: current evidence and perspectives.

Ducreux B, Patrat C, Firmin J, Ferreux L, Chapron C, Marcellin L Clin Epigenetics. 2025; 17(1):32.

PMID: 39985111 PMC: 11846336. DOI: 10.1186/s13148-025-01828-w.

Assessment of candidate high-grade serous ovarian carcinoma predisposition genes through integrated germline and tumour sequencing.

Subramanian D, Zethoven M, Pishas K, Marinovic E, McInerny S, Rowley S NPJ Genom Med. 2025; 10(1):1.

PMID: 39794353 PMC: 11724014. DOI: 10.1038/s41525-024-00447-3.

Multiplex digital profiling of DNA methylation heterogeneity for sensitive and cost-effective cancer detection in low-volume liquid biopsies.

Zhao Y, OKeefe C, Hu J, Allan C, Cui W, Lei H Sci Adv. 2024; 10(47):eadp1704.

PMID: 39576863 PMC: 11584010. DOI: 10.1126/sciadv.adp1704.

COL25A1 and METAP1D DNA methylation are promising liquid biopsy epigenetic biomarkers of colorectal cancer using digital PCR.

Overs A, Peixoto P, Hervouet E, Molimard C, Monnien F, Durand J Clin Epigenetics. 2024; 16(1):146.

PMID: 39425144 PMC: 11490026. DOI: 10.1186/s13148-024-01748-1.

Amplicon-Based Bisulfite Conversion-NGS DNA Methylation Analysis Protocol.

Albrecht C, Bashtrykov P, Jeltsch A Methods Mol Biol. 2024; 2842:405-418.

PMID: 39012608 DOI: 10.1007/978-1-0716-4051-7_21.

References

Gruenbaum Y, Stein R, Cedar H, Razin A . Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 1981; 124(1):67-71. DOI: 10.1016/0014-5793(81)80055-5. View

Bird A . Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern. J Mol Biol. 1978; 118(1):49-60. DOI: 10.1016/0022-2836(78)90243-7. View

Adouard V, Dante R, Niveleau A, Delain E, Revet B, Ehrlich M . The accessibility of 5-methylcytosine to specific antibodies in double-stranded DNA of Xanthomonas phage XP12. Eur J Biochem. 1985; 152(1):115-21. DOI: 10.1111/j.1432-1033.1985.tb09170.x. View

Gardiner-Garden M, Frommer M . CpG islands in vertebrate genomes. J Mol Biol. 1987; 196(2):261-82. DOI: 10.1016/0022-2836(87)90689-9. View

Altschul S, Gish W, Miller W, Myers E, Lipman D . Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10. DOI: 10.1016/S0022-2836(05)80360-2. View

Frommer M, McDonald L, Millar D, Collis C, Watt F, GRIGG G . A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992; 89(5):1827-31. PMC: 48546. DOI: 10.1073/pnas.89.5.1827. View

Clark S, Harrison J, Paul C, Frommer M . High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994; 22(15):2990-7. PMC: 310266. DOI: 10.1093/nar/22.15.2990. View

Raizis A, Schmitt F, Jost J . A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal Biochem. 1995; 226(1):161-6. DOI: 10.1006/abio.1995.1204. View

Herman J, Graff J, Myohanen S, Nelkin B, Baylin S . Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996; 93(18):9821-6. PMC: 38513. DOI: 10.1073/pnas.93.18.9821. View

10.

Xiong Z, Laird P . COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997; 25(12):2532-4. PMC: 146738. DOI: 10.1093/nar/25.12.2532. View

11.

Singal R, Ginder G . DNA methylation. Blood. 1999; 93(12):4059-70. View

12.

Singal R, Grimes S . Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction. Biotechniques. 2001; 30(1):116-20. DOI: 10.2144/01301bc02. View

13.

Robertson K, Wolffe A . DNA methylation in health and disease. Nat Rev Genet. 2001; 1(1):11-9. DOI: 10.1038/35049533. View

14.

Kampke T, Kieninger M, Mecklenburg M . Efficient primer design algorithms. Bioinformatics. 2001; 17(3):214-25. DOI: 10.1093/bioinformatics/17.3.214. View

15.

Grunau C, Clark S, Rosenthal A . Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 2001; 29(13):E65-5. PMC: 55789. DOI: 10.1093/nar/29.13.e65. View

16.

Gitan R, Shi H, Chen C, Yan P, Huang T . Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res. 2002; 12(1):158-64. PMC: 155260. DOI: 10.1101/gr.202801. View

17.

Warnecke P, Stirzaker C, Song J, Grunau C, Melki J, Clark S . Identification and resolution of artifacts in bisulfite sequencing. Methods. 2002; 27(2):101-7. DOI: 10.1016/s1046-2023(02)00060-9. View

18.

Li L, Dahiya R . MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002; 18(11):1427-31. DOI: 10.1093/bioinformatics/18.11.1427. View

19.

Humeny A, Beck C, Becker C, Jeltsch A . Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal Biochem. 2003; 313(1):160-6. DOI: 10.1016/s0003-2697(02)00568-7. View

20.

Couvert P, Poirier K, Carrie A, Chalas C, Jouannet P, Beldjord C . DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes. Biotechniques. 2003; 34(2):356-62. DOI: 10.2144/03342rr06. View