TY - JOUR
T1 - A novel method to capture methylated human DNA from stool
T2 - Implications for colorectal cancer screening
AU - Zou, Hongzhi
AU - Harrington, Jonathan
AU - Rego, Rafaela L.
AU - Ahlquist, David A.
PY - 2007/9
Y1 - 2007/9
N2 - 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.
AB - 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.
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U2 - 10.1373/clinchem.2007.086223
DO - 10.1373/clinchem.2007.086223
M3 - Article
C2 - 17712002
AN - SCOPUS:34548354511
SN - 0009-9147
VL - 53
SP - 1646
EP - 1651
JO - Clinical chemistry
JF - Clinical chemistry
IS - 9
ER -