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Title: Identification of highly methylated human DNA sequences using a methyl-CpG binding domain column
Author: Brock, Graham J. R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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A library of densely methylated sequences has been constructed from DNA derived from human blood. Genomic DNA was fractionated using a column which binds preferentially according to methyl-CpG density. The library contains known fragments of methylated CpG Islands (CGIs) and other novel regions with a high density of m5CpG. Analysis of cloned inserts demonstrated that most were more GC-rich and had a higher GpGObs/Exp than bulk genomic DNA. Cloned inserts were used as probes against southern blots of DNA digested with methylation sensitive or insensitive isoschisomers. The hybridisation patterns demonstrated that majority of the cloned sequences tested were methylated in DNA when derived from blood. One of the most frequently occurring clones in the library matched to a fragment from the rDNA repeat non transcribed spacer (NTS). This entire region has subsequently been shown to be heavily methylated in DNA from somatic cells. The cloned fragments derived from both male and female DNA were used as probes in Fluorescent in situ Hybridisation (FISH) experiments. DNA derived from both male and female gave indistinguishable hybridisation patterns with many fragments apparently hybridising to subtelomeric regions. The other regions of significant hybridisation were to the short arms of the acrocentrics an the centromere of chromosome 9. The rDNA repeats are located on the short arms of the acrocentric chromosomes while the centromere of chromosome 9 has been reported to contain low copy number methylated repeats. These sequences though methylated, in DNA from somatic cells, are often unmethylated in sperm. This apparent lack of germ-line methylation may account for the high frequency of CpG in these regions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available