18 Quantitative DNA Fiber Mapping
245
formed on images recorded on film and either printed or projected on a
screen.
Procedure
Images are acquired using a standard fluorescence microscope (Zeiss
Axioskop) equipped with 63x, 1.25 N.A. and 40x, 1.2 N.A. objectives,
and a filter set for excitation and simultaneous observation ofDAPI, Texas
Red/rhodamine, FITC and CY5 fluorescence, respectively (ChromaTechnology). Current filters are capable of excitation in single bands centered around 360, 405, 490, 555, and 637 nm, and visualization in multiple
bands in the vicinities of 460 nm (blue), 520 nm (green), 600 nm (red) and
680 nm (infrared). Images are collected using a CCD camera (Xilix, Hamamatsu or Photometrics) connected to a computer workstation (Weier
et aI., 1995).
For determination of map positions, interactive software is available
for either Apple Macintosh, IBM/PC or SUN computers that allows the
user to trace DNA fibers by drawing a segmented line and then calculates
the length of the line in pixels (Wang et ai. 1996; Duell et ai. 1997). The
pixel spacing is known from the microscope objective used in the experiment (use a 63x objective for molecules up to 100 kb, a 40x objective for
larger molecules) and is converted into ~m (or kb using the factor of 2.3
kb/~m). After measuring all relevant distances along the DNA fibers in
triplicate, the results in the form oflists are imported into Microsoft Excel
spreadsheets and used to calculate average values for each fiber and mean
values and standard deviations for individual experiments.
Results
Solid substrates for QDFM are prepared in batches of 20-50 by derivatization of standard microscope slides, coverslips or sheets of mica with APS,
which results in primary amino groups on the surface (Weier et ai. 1995;
Hu et ai. 1996). For DNA fiber stretching, a solution of target DNA molecules onto which probes are to be mapped is placed on an untreated
coverslip and spread by placing the coverslip upside-down on the APSderivatized glass or mica surface. Binding of DNA to the substrate and
the stretching effect can be monitored by staining the DNA with
YOYO-l prior to deposition. This also allows the rejection of batches
of slides that bind DNA too tightly. Following DNA binding and stretch-
245
formed on images recorded on film and either printed or projected on a
screen.
Procedure
Images are acquired using a standard fluorescence microscope (Zeiss
Axioskop) equipped with 63x, 1.25 N.A. and 40x, 1.2 N.A. objectives,
and a filter set for excitation and simultaneous observation ofDAPI, Texas
Red/rhodamine, FITC and CY5 fluorescence, respectively (ChromaTechnology). Current filters are capable of excitation in single bands centered around 360, 405, 490, 555, and 637 nm, and visualization in multiple
bands in the vicinities of 460 nm (blue), 520 nm (green), 600 nm (red) and
680 nm (infrared). Images are collected using a CCD camera (Xilix, Hamamatsu or Photometrics) connected to a computer workstation (Weier
et aI., 1995).
For determination of map positions, interactive software is available
for either Apple Macintosh, IBM/PC or SUN computers that allows the
user to trace DNA fibers by drawing a segmented line and then calculates
the length of the line in pixels (Wang et ai. 1996; Duell et ai. 1997). The
pixel spacing is known from the microscope objective used in the experiment (use a 63x objective for molecules up to 100 kb, a 40x objective for
larger molecules) and is converted into ~m (or kb using the factor of 2.3
kb/~m). After measuring all relevant distances along the DNA fibers in
triplicate, the results in the form oflists are imported into Microsoft Excel
spreadsheets and used to calculate average values for each fiber and mean
values and standard deviations for individual experiments.
Results
Solid substrates for QDFM are prepared in batches of 20-50 by derivatization of standard microscope slides, coverslips or sheets of mica with APS,
which results in primary amino groups on the surface (Weier et ai. 1995;
Hu et ai. 1996). For DNA fiber stretching, a solution of target DNA molecules onto which probes are to be mapped is placed on an untreated
coverslip and spread by placing the coverslip upside-down on the APSderivatized glass or mica surface. Binding of DNA to the substrate and
the stretching effect can be monitored by staining the DNA with
YOYO-l prior to deposition. This also allows the rejection of batches
of slides that bind DNA too tightly. Following DNA binding and stretch-