Single molecule analysis of DNA replication

Biochimie. Aug-Sep 1999;81(8-9):859-71. doi: 10.1016/s0300-9084(99)00210-2.

Abstract

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • DNA Replication / genetics
  • DNA Replication / physiology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Female
  • Gene Amplification
  • Gene Rearrangement
  • Genome
  • In Situ Hybridization, Fluorescence
  • Kinetics
  • Male
  • Molecular Biology / methods*
  • Replication Origin
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Surface Properties
  • Xenopus laevis

Substances

  • DNA