Application of DNA micromanipulation to gene mapping and localized restriction enzyme digestion

Accurate manipulation of a single DNA molecule will support genome analysis, such as gene mapping, physical mapping and sequencing of chromosome. This paper presents the micromanipulation of single-stranded DNA and its application to gene mapping. As a model template DNA, monomeric λ phage DNA was tandemly ligated which was then denatured and hybridized with a cloned probe DNA of λ-exo gene region. The hybridized molecules were terminally bound with magnetic beads, elongated, and video-imaged. We could observe the islands of gleaming spots which stood in line from a bead at regular intervals. This agree well with the expected images from the construction of the templates and probes. We have also demonstrated the localization of restriction enzyme activity only in a small area of stretched long chromosomal DNA using temperature control technique. The DNAs were digested about 10 μm around the laser focal spot (about 10 pico liter portion) under a microscopic observation view. This technique may open up quick physical mapping by handling a single DNA molecule