Abstracts: 



Mapping 



Instrumentation 



Thermal Stability Mapping of DN A by Random 

 Fragmentation and Two-Dimensional Denaturing 

 Gradient Electrophoresis 



L. S. Lerman. Nashua Gabra, Eric Schmitt. and Ezra Abrams 



Department of Biology, Massachusetts Institute of Technology. Cambridge. MA 02139 



(617)253-6658 



The themial stability of the double helix in a standard solvent is fully determined by the 

 base sequence. Within a long DNA molecule, each local region (ranging in length from 

 a few dozen bases up to several hundred ba.se pairs) undergoes a transition from an 

 ordered helix to a disordered, randomized configuration (melting) within a narrow 

 temperature span, typically from I to 3°C for the change from 95*"/^ helical to 5% 

 helical. It is convenient to characterize the transition in each region, or domain, by the 

 Tm, the temperature at which there is a 50-50 equilibrium between the helical and 

 melted forms. Within human genomic DNA there is substantial variation in the local 

 Tm, as much as about 35 degrees, often with distinct and sharp boundaries between 

 adjacent domains. While the pattern and characteristics of this sequence of domains in 

 long DNA molecules is inferred principally by statistical-mechanical theory, the domain 

 content is more directly observable in short DNA molecules by means of absorption 

 spectroscopy as a function of temperature, or by denaturing gradient electrophoresis. 

 Since the Tm of each domain is changed only very slightly by the substitution, addition, 

 or deletion of one or a very few bases, the sequence of domains provides a robust 

 counterpart to the base sequence. The domain map is less sensitive to trivial individual 

 variation (including methylation) than a restriction map and reflects biological function 

 more closely. 



In two-dimensional separation of randomly fragmented genomic DNA. each random 

 fragment is identified by X. Y coordinates representing its length and the Tm of the 

 domain with the lowest Tm in the fragment. All fragments in which that domain is the 

 lowest will find a similar gradient level, regardless of their length. This distribution and 

 the response to specific sequence probes provide a means, in principle, for determining 

 the spacing, order, and Tm among those domains that have a lower Tm than the average 

 and those with the highest Tm. It will provide measurements of the nucleotide distances 

 between each of these domains and any arbitrary set of sequence identifiers or probes. 



Our current effort is concerned with refining and calibrating various aspects of the two- 

 dimensional denaturing gradient technique and related procedures. The.se efforts include 

 (I) using iron-peroxide nicking and SI cleavage for the preparation of fully random 

 distribution of fragments from lambda DNA and yeast artificial chromosomes 

 containing long human genomic inserts; (2) reducing the breadth of bands produced by 

 very long DNA molecules in the denaturing gradient: (3) analyzing the band broadening 

 observed when the domain with lowest Tm is surrounded by higher melting domains; 

 and (4) developing optical, mathematical, and computing procedures for calibrating gel 

 photographs or autoradiographs in terms of quantitative, point-to-point distributions of 

 DNA. 



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