A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening.
BACKGROUND
Assay of methylated DNA markers in stool is a promising approach for colorectal cancer (CRC) screening. A method to capture hypermethylated CpG islands from stool would enrich target analyte and allow optimal assay sensitivity.


METHODS
Methyl-binding domain (MBD) protein was produced using a pET6HMBD plasmid with MBD DNA sequence cloned from rat MeCP2 gene and bound to a column of nickel-agarose resin. We first established the feasibility of using the MBD column to extract methylated human DNA in a high background of fecal bacterial DNA. To explore the impact of MBD enrichment on detection sensitivity, the tumor-associated methylated vimentin gene was assayed with methylation-specific PCR from stools to which low amounts of cancer cell DNA (0-50 ng) were added and from stools from CRC patients and healthy individuals. Stools from cancer patients were selected with low amounts of human DNA (median 7 ng, range 0.5-832 ng).


RESULTS
With MBD enrichment, methylated vimentin was detected in stools enriched with >/=10 ng of cancer cell DNA and in CRC stool with a range of native human DNA amounts from 4 to 832 ng. Without MBD enrichment, methylated vimentin was not detected in the enriched stools and was detected in only 1 cancer stool with high human DNA (832 ng). In stools from healthy individuals methylated vimentin was not detected, with or without MBD enrichment.


CONCLUSIONS
MBD capture increases assay sensitivity for detecting methylated DNA markers in stool. Applied clinical studies for stool cancer screening are indicated.
Zou
Hongzhi
H
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
Harrington
Jonathan
J
Rego
Rafaela L
RL
Ahlquist
David A
DA
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis*; D018899:CpG Islands; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D005240:Feasibility Studies; D005243:Feces / Q000737:chemistry*; D005819:Genetic Markers; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D051381:Rats; D012680:Sensitivity and Specificity; D014746:Vimentin / Q000032:analysis* / Q000235:genetics
D014408:Biomarkers, Tumor; D004269:DNA, Bacterial; D004273:DNA, Neoplasm; D005819:Genetic Markers; C497394:Mecp2 protein, rat; D051783:Methyl-CpG-Binding Protein 2; D014746:Vimentin