A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples
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The bisulfite genomic sequencing (BGS) protocol (1 , 2 ) is a method of choice for analyzing DNA methylation at the nucleotide level. Sodium bisulfite is used to convert unmethylated cytosine residues to uracil residues in single-stranded DNA. In particular, bisulfite conversion consists of three sequential chemical reactions: sulfonation of cytosine to cytosine-6-sulfonate, deamination to uracil-6-sulfonate, and desulfonation to uracil. However, since 5-methylcytosine residues are nonreactive, they remain intact. The bisulfite-converted DNA is then amplified with specific primers designed for converted DNA, and purified polymerase chain reaction (PCR) products, which are usually subcloned, are sequenced.
Bisulfite conversion is so powerful that it has been paired with numerous techniques other than traditional sequencing, including: methylation-specific PCR (3 ), combined bisulfite restriction enzyme analysis (4 ), methylation-sensitive single nucleotide primer extension (5 ), methylation-sensitive single-strand conformation analysis (6 ), MethyLight (7 ), oligonucleotide microarray methods (8 ), denaturing high-performance liquid chromatography with bisulfite genomic sequencing (9 ), pyrosequencing methylation analysis (10 ), and methylation-sensitive high-resolution melting-curve analysis (11 ), among others (see (12 ) for a review). In addition, many methylation analysis kits are also commercially available.
Unfortunately, high rates of DNA degradation and incomplete conversion reactions often lead to decreased efficiency of the assay. Many attempts have been made to minimize template degradation and/or maximize cytosine conversion (13 � 19 ), but overall, the bisulfite conversion protocol has remained unchanged, and no other high resolution or positive display methylation analysis protocol exists. As a result, the BGS protocol, as well as any technique paired with the bisulfite conversion reaction (and, hence, founded on the assumption that conversion is complete) often generate few or no informative results.
In our studies of the RARB2 P2 promoter (20 ), we found that incomplete conversion was an insurmountable challenge even after modifying the protocol in numerous ways. We, therefore, aimed to circumvent these issues altogether by depleting the PCR populations of products amplified from partially converted or unconverted DNA using a multiple restriction enzyme digestion (MRED) approach. We found that informative sequencings were increased ninefold using it. We believe that this method may easily be adapted for analyzing the detailed methylation status of other genes presenting incomplete cytosine to uracil conversion, and we provide guidelines for selecting the most appropriate restriction enzymes (REs).
Cell-Line Provenance
Twenty-one cell lines were cultured. CALU-1, SK-MES, CACO-2, COLO-201, COLO-205, HCT-15, and LS-180 were obtained from the American Type Culture Collection (Rockville, MD). The CALU-1 daughter cell lines, C-19 and C-59, are RARB2 -transfectants that were established in our laboratory (21 ). MM-1 was also established in our laboratory (6 ). NCI-H23, NCI-H82, NCI-H125, NCI-H157, NCI-H520, and NCI-H596 were supplied by Dr. Adi Gazdar (NCI, NIH, Bethesda, MD). NBE-E6 E7 (22 ) was provided by Dr. Jean Viallet (Gemin X Biotechnologies Inc., Montreal, Québec). SW 1222 was given to us by Dr. Clifford Stanners (McGill University, Montreal, Québec). Qu-DB was provided by Dr. Barbara Campling (Queen’s University, Kingston, Ontario). T47D, MDA-MB-231 (MB-231), ZR-75B, and HS-578T were kindly provided by Dr. Morag Park (McGill University, Montreal, Québec).
Cell Culture
CALU-1, CACO-2, SW-1222, and LS-180 were grown in α-MEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Wisent Bioproducts, Saint-Jean-Baptiste de Rouville, Québec). NBE-E6 E7 was grown in keratinocyte-serum free medium (Invitrogen), supplemented with 50 µg/ml bovine pituitary extract, and 5 ng/ml recombinant human epidermal growth factor (Invitrogen). All other cells were grown in RPMI-1640 medium (Invitrogen) supplemented either with 5% (SK-MES, NCI-H23, NCI-H125, NCI-H520, Qu-DB, and HS-578T) or 10% FCS (NCI-H82, NCI-H157, MM-1, T47D, MDA-MB-231, ZR-75B, COLO-201, COLO-205, and HCT-15). Where indicated, cells were treated with 1 µM 5-azadeoxycytidine.
Genomic DNA Extraction