Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA Page: 4 of 30
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circumvented [11,19]. Mechanical deposition of DNA molecules, however, does not
allow precise control of the extent of DNA stretching so that molecules are often
stretched to various extents, and standards are needed for normalization and distance
measurements [11,17,20]. Results from our and other laboratories suggest that
stretching of DNA molecules bound to a solid substrate at one or both of their respective
ends by the hydrodynamic action of a receding water meniscus termed 'molecular
combing'  produces linear DNA molecules that are stretched homogeneously to
approximately 2.3kb/pm [18,22]. We have applied this procedure to prepare linear DNA
molecules ('DNA fibers') ranging in size from 5-6kb to more than 1000kb for localization
of restriction fragments, cloned DNA or cDNA sequences along the fibers or for
measurement of physical distances between non-overlapping DNA molecules
[3,4,23,24]. To emphasize the quantitative character of this optical mapping procedure
comprised of molecular combing, FISH, and digital image analysis, we termed the
process 'Quantitative DNA Fiber Mapping' or 'QDFM' [18,22,23].
Gene mapping and construction of high-resolution physical maps constitute major
applications of QDFM in our laboratory. For these purposes, large insert DNA clones
are obtained from the Dupont P1 library by screening [25,26]. As practiced previously
, QDFM on the P1 clones required separation of high molecular weight DNA from
the bacterial host, linearization of the circular P1 DNA molecules by restriction in the
unique Not1 site within the P1 vector pSac BII, and PFGE purification of the linear P1
DNA molecules. The linearized P1 molecules were then bound to silanated glass slides,
subjected to molecular combing, and used as mapping templates [23,27].
Occasionally, we encounter clones that are smaller than the 'standard' P1 clones
of ~95kb. These clones typically yield less DNA in our alkaline lysis protocol , and
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Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F. & Weier, Heinz-Ulli G. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA, report, December 4, 2008; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1013868/m1/4/: accessed November 18, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.