TY - JOUR
T1 - DNA methylation mapping by tag-modified bisulfite genomic sequencing
AU - Han, Weiguo
AU - Cauchi, Stephane
AU - Herman, James G.
AU - Spivack, Simon D.
N1 - Funding Information:
At the Wadsworth Center, the authors thank Shalini Kumar for organizational laboratory support, Dr. Laurence Kaminsky for administrative laboratory support, Matt Schudt and the Molecular Genetics Core for technical support in sequencing, the Biochemistry Core, and Dr. Adriana Verschoor for manuscript editing. At Albany Medical Center, research nurses Angela Sheehan, Kathy Mokhiber, and Ann Venezia contributed exemplary subject recruitment, biospecimen collection, and organizational skills. Dr. Riivo Ilves, Thoracic Surgery and Dr. Tim Jennings, Anatomic Pathology, facilitated the procurement of surgical lung tissue, and clinical colleagues in Pulmonary & Critical Care Medicine kindly permitted the enrollment of their patients. This study was supported by NIH-R21 CA 94714 (to SDS), and NIH-R21 CA 10481 (to SDS), and NIH-R01 CA 106186 (to SDS).
PY - 2006/8/1
Y1 - 2006/8/1
N2 - A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R = 0.935, p = 0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p < 0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.
AB - A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R = 0.935, p = 0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p < 0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.
KW - Bisulfite genomic sequencing
KW - CYP1B1
KW - DNA methylation
KW - Exfoliated cells
KW - GSTP1
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U2 - 10.1016/j.ab.2006.05.010
DO - 10.1016/j.ab.2006.05.010
M3 - Article
C2 - 16797472
AN - SCOPUS:33745914971
SN - 0003-2697
VL - 355
SP - 50
EP - 61
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -