Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation.
DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the "gold standard" for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.
Reed
Kerry
K
Biomolecular Sciences Program, Laurentian University, Sudbury, Ont. P3E 2C6, Canada.
Poulin
Matthew L
ML
Yan
Liying
L
Parissenti
Amadeo M
AM
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D013447:Sulfites; C042345:hydrogen sulfite