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Clin Chem. 2007 Sep;53(9):1646-51.

A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening.

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  • 1Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.

Abstract

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.

PMID:
17712002
DOI:
10.1373/clinchem.2007.086223
[PubMed - indexed for MEDLINE]
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