An AscI Boundary Library for the Studies of Genetic and Epigenetic Alterations in CpG Islands (original) (raw)

  1. Zunyan Dai1,2,
  2. Dieter Weichenhan4,
  3. Yue-Zhong Wu1,
  4. Julia L Hall1,
  5. Laura J. Rush1,3,
  6. Laura T. Smith1,
  7. Aparna Raval1,
  8. Li Yu1,
  9. Daniela Kroll5,
  10. Joerg Muehlisch5,
  11. Michael C. Frühwald5,
  12. Pieter de Jong6,
  13. Joe Catanese6,
  14. Ramana V. Davuluri1,
  15. Dominic J. Smiraglia1, and
  16. Christoph Plass1,7
  17. 1Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics,2Department of Pathology, 3Department of Veterinary Biosciences, and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA; 4Medizinische Universität zu Lübeck, Medizinische Klinik II, Ratzeburger Allee 160 23538 Lübeck, Germany; 5Klinik und Poliklinik für Kinderheilkunde, Pädiatrische Hämatologie/Onkologie, Universitätsklinikum Münster, 48149 Münster, Germany; 6Children's Hospital, Oakland Research Institute, Oakland, California 94609, USA

Abstract

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using_Not_I as the restriction landmark site. Here, we describe the development of RLGS using _Asc_I as the restriction landmark site for genome-wide scans of cancer genomes. The availability of _Asc_I as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using _Not_I only. We describe the development of an _Asc_I–_Eco_RV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 _Asc_I RLGS fragments via two methods by use of this library.

[Supplemental material is available online at http://www.genome.org.]

Footnotes