title: COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.
abstract: Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Castellanos-Rizaldos
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Coren A
authors.initials: CA
authors.lastname: Milbury
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Elli
authors.initials: E
authors.lastname: Karatza
authors.affiliation: Division of Neurosurgery, University of California San Diego, San Diego, California, United States of America.
authors.forename: Clark C
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America; Division of Medical Physics and Biophysics, Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
authors.affiliation: Division of General Pediatrics, Boston Medical Center and Boston University, Boston, Massachusetts, United States of America.
authors.forename: Anne
authors.initials: A
authors.lastname: Merewood
mesh_terms: D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
chemical_list: D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite
references.citation: Oncogene. 2002 Aug 12;21(35):5400-13
references.pmid: 12154403
references.citation: Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827-31
references.pmid: 1542678
references.citation: Clin Chem. 2009 Dec;55(12):2130-43
references.pmid: 19815609
references.citation: Arch Med Res. 2013 May;44(4):281-90
references.pmid: 23608672
references.citation: PLoS Genet. 2013 Nov;9(11):e1003873
references.pmid: 24278026
references.citation: Nat Med. 2008 May;14(5):579-84
references.pmid: 18408729
references.citation: Hum Mutat. 2010 Dec;31(12):1366-73
references.pmid: 20848649
references.citation: Nucleic Acids Res. 2011 Jan;39(1):e2
references.pmid: 20937629
references.citation: Mol Cell Biol. 2003 Aug;23(15):5293-300
references.pmid: 12861015
references.citation: Endocrinology. 2013 Sep;154(9):3476-81
references.pmid: 23825129
references.citation: J Mol Diagn. 2010 Sep;12(5):705-11
references.pmid: 20616366
references.citation: Biochem Biophys Res Commun. 2006 Jun 9;344(3):821-6
references.pmid: 16631604
references.citation: J Mol Diagn. 2011 Mar;13(2):220-32
references.pmid: 21354058
references.citation: Nucleic Acids Res. 2007;35(6):e41
references.pmid: 17289753
references.citation: Clin Chem. 2007 Dec;53(12):2119-27
references.pmid: 17962364
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6
references.pmid: 8790415
references.citation: Biochem Soc Trans. 2009 Apr;37(Pt 2):427-32
references.pmid: 19290875
references.citation: Nucleic Acids Res. 2000 Apr 15;28(8):E32
references.pmid: 10734209
references.citation: Cancer Res. 2000 Nov 1;60(21):5954-8
references.pmid: 11085511
references.citation: J Pathol. 2007 Feb;211(3):261-8
references.pmid: 17177177
references.citation: Hum Mutat. 2009 Nov;30(11):1583-90
references.pmid: 19760750
references.citation: Nucleic Acids Res. 1997 Jun 15;25(12):2532-4
references.pmid: 9171110
references.citation: Expert Rev Mol Diagn. 2011 Mar;11(2):159-69
references.pmid: 21405967
references.citation: Lancet Oncol. 2009 May;10(5):459-66
references.pmid: 19269895
references.citation: Clin Chem. 2010 Aug;56(8):1287-96
references.pmid: 20551384
references.citation: Horm Res. 2009 Jan;71 Suppl 1:13-6
references.pmid: 19153498
references.citation: BMC Clin Pathol. 2010 Nov 26;10:6
references.pmid: 21110880