An AscI Boundary Library for the Studies of Genetic and Epigenetic Alterations in CpG Islands

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 NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI–EcoRV 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 AscI RLGS fragments via two methods by use of this library.[Supplemental material is available online at http://www.genome.org.]

Epigenetic Profiling in Chronic Lymphocytic Leukemia Reveals Novel Methylation Targets

“…The procedure for the AscI-EcoRV-HinfI enzyme combination is essentially the same except that DNA is first digested with EcoRV followed by the methylation-sensitive enzyme AscI (recognition site GG2CGCGCC; Ref. 28).…”

“…RLGS loci that were present in control tissue profiles but absent in CLL profiles were recorded for each CLL profile using our "master profile" recording system published previously (10,28). Each locus on the NotI-EcoRV-HinfI and AscI-EcoRV-HinfI profiles has been assigned a unique address consisting of a row and column (delineating a section on the profile) in addition to a unique number within that section.…”

Aberrant DNA Methylation of OLIG1, a Novel Prognostic Factor in Non-Small Cell Lung Cancer

“…RLGS was performed using both NotI and AscI as restriction landmark enzymes. As previously reported [10], the recognition sequences of these enzymes occur preferentially within CpG islands as defined by Gardiner-Garden and Frommer [28], effectively creating a bias towards the assessment of DNA methylation in promoter sequences [15]. Additionally, recent bioinformatics analyses indicate that 92.7% of NotI sites fall within the 5′ end, inside, or 3′ end of transcripts (R.V.…”

“…RLGS fragments of interest that had not already been identified in our laboratory were cloned with the aid of either a human NotI–EcoRV or a human AscI–EcoRV plasmid library, as previously described [13,15,16]. Alternatively, a PCR-based approach was employed to identify RLGS fragments not present in the libraries [16].…”

Translating insights from the cancer genome into clinical practice

“…First, restriction-landmark genomic scanning 84 using methylation-sensitive enzymes was the first method developed as a genome-wide screen for methylation of CpG islands. This technique, which involves two restriction digests followed by electrophoretic separation, allows methylation to be analysed in up to 4,000 loci 85,86 . Second, microarray-based epigenomic analysis methods 87 involve hybridization of tumour and reference DNA samples to DNA microarrays.…”