Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing
DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA–protein interaction, and DNA methylation along genomic single DNA molecules at 1 kb resolution. However, DNA combing has been restricted to short 200–500 kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.
Key wordsDNA combing Single DNA molecules Fluorescent in situ hybridization Immunodetection Replication origin firing Replication fork velocity Genomic rearrangements
This work was supported by Irma T. Hirschl and Charles Revson postdoctoral fellowships and Wellcome Trust Grant to PN [grant number 093917] and The Breast Cancer Research Foundation.
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