18 Quantitative DNA Fiber Mapping
237
determined primarily by the DNA preparation and by properties of the
modified surface rather than by the method of DNA stretching. In general,
the ideal APS-surface binds the DNA molecules only at their ends, or in the
case of circular DNA molecules, at the position of nicks. The remainder of
the DNA molecule should be free in suspension. This can be observed in
the fluorescence microscope after addition of 1 IlM YOYO-l to the DNA
before immobilization.
Procedure
In a typical experiment, 1-2 III of clonal or genomic DNA are mixed with
an equal amount of YOYO-l (lIlM or O.IIlM) and 8 III water. One or two
microliter of this diluted DNA is applied to an untreated coverslip, which
is then placed DNA side down on the APS-treated slide or coverslip. The
DNA concentration can be estimated under the fluorescence microscope
using a filter set for FITC, and adjusted as needed. As early as 2 min of
incubation at room temperature, the untreated coverslip can be removed
slowly from one end, allowing the receding meniscus to stretch the bound
DNA molecules ('fibers') in one direction (Hu et aI., 1996). Alternatively,
the slide or coverslip sandwich can be allowed to dry overnight at room
temperature, after which the untreated coverslip is removed. Slides or coverslips carrying DNA fibers are rinsed briefly with water, drained, allowed
to dry at room temperature and 'aged' in ambient air at 20°C for 1 week
before hybridization.
Subprotocol 4
Probes generated from cloned DNA fragments
A typical QDFM experiment uses several different probes simultaneously.
One probe is needed to counterstain the DNA fibers. This probe is usually
prepared by labeling DNA from the same batch that was used to prepare
the fibers. Probes for sequences to be mapped along the fibers are made
such that they can be detected in a different color. Furthermore, it is recommended to include landmark probes that provide reference points by
binding specifically to the vector part or the ends of DNA molecules.
237
determined primarily by the DNA preparation and by properties of the
modified surface rather than by the method of DNA stretching. In general,
the ideal APS-surface binds the DNA molecules only at their ends, or in the
case of circular DNA molecules, at the position of nicks. The remainder of
the DNA molecule should be free in suspension. This can be observed in
the fluorescence microscope after addition of 1 IlM YOYO-l to the DNA
before immobilization.
Procedure
In a typical experiment, 1-2 III of clonal or genomic DNA are mixed with
an equal amount of YOYO-l (lIlM or O.IIlM) and 8 III water. One or two
microliter of this diluted DNA is applied to an untreated coverslip, which
is then placed DNA side down on the APS-treated slide or coverslip. The
DNA concentration can be estimated under the fluorescence microscope
using a filter set for FITC, and adjusted as needed. As early as 2 min of
incubation at room temperature, the untreated coverslip can be removed
slowly from one end, allowing the receding meniscus to stretch the bound
DNA molecules ('fibers') in one direction (Hu et aI., 1996). Alternatively,
the slide or coverslip sandwich can be allowed to dry overnight at room
temperature, after which the untreated coverslip is removed. Slides or coverslips carrying DNA fibers are rinsed briefly with water, drained, allowed
to dry at room temperature and 'aged' in ambient air at 20°C for 1 week
before hybridization.
Subprotocol 4
Probes generated from cloned DNA fragments
A typical QDFM experiment uses several different probes simultaneously.
One probe is needed to counterstain the DNA fibers. This probe is usually
prepared by labeling DNA from the same batch that was used to prepare
the fibers. Probes for sequences to be mapped along the fibers are made
such that they can be detected in a different color. Furthermore, it is recommended to include landmark probes that provide reference points by
binding specifically to the vector part or the ends of DNA molecules.