DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory.

Abstract

DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring highermelting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition *of mutants in the next ranking domain.Detection .and localization of single base substitutions within long DNA sequences may be impractical by complete sequence determination and improbable on the basis of restriction endonuclease vulnerability. We present here the results of a procedure by which DNA molecules that have minimal sequence differences are separated and by which some conclusions can be drawn as to the nature of the change. A number of samples can conveniently be examined in a single slab gel; each DNA species is focused into a sharp band-at a gel position determined by its sequence and composition. The physical separation of fragments of altered sequence provided by the denaturing gel makes possible further analysis and manipulation.Our system makes an unconventional use of electrophoresis. Where DNA molecules migrate into a gradient of ascending concentration of denaturant, they undergo an -abrupt decrease in mobility at a characteristic depth, resulting in positions and patterns that change. little if application of the field is continued. The retardation depth in the gradient is determined by the least stable part of the molecule and is relatively insensitive to other parts of the sequence or to the overall length (1).To understand the basis of the' sensitivity of the system to single base substitutions, we have undertaken a close comparison of the gel results with those of a sequence-specific statistical mechanical theory of the stability of the double helix. Experimental studies on the helix-disorder transition, melting,have not yet provided a detailed test of the theory, which pre-'dicts intricate and interesting patterns for the progression of equilibria from full helicity to separated strands as the temperature increases for molecules of different sequence. In our system, the molecule is exposed to a gradual denaturation-promoting change in the medium, linearly equivalent (2) to a gradual increase in temperature. 'The strong decrease in mobility as the helix unravels provides the basis for...

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

“…In the 1980s multiple techniques were developed that screened samples for single base variants at specific, PCR-amplified loci[22, 23]. Examples include denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), which are based on the exquisite sensitivity of DNA denaturation for sequence variants; two 500-bp fragments of similar size, differing in only one base pair melt at different temperatures and can be separated by gel electrophoresis in a gradient of chemical denaturants or temperature[24–26]. The sensitivity of these assays can be greatly increased by first allowing a mixture of mutant and wildtype fragments to denature and then slowly reanneal.…”

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

“…These enzymes (mostly 4-bp cutters) usually produce small (200-700 bp) DNA fragments that contain at least two melting domains. Since DGGE can reveal differences in all but the highest temperature (most stable) melting domain of a DNA fragment, the use of frequent cutting restriction enzymes is best for DGGE analysis (Fischer and Lerman, 1983;Abrams and V.P. Stanton, 1992).…”

“…Consequently, both DNA sequence polymorphisms and methylation differences in purified DNA fragments have been revealed by DGGE. Since detection by DGGE does not rely on the modified base occurring within a restriction enzyme recognition site, methylated nucleotides can be detected indiscriminately if the modifications occur within the DNA sequence of the least stable melting domain of the DNA fragment (Fischer and Lerman, 1983;Abrams and V.P. Stanton, 1992).…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…The improved methods have included modes of sequencing DNA [5][6][7] , allele-specific recognition of mutant sequences [8][9][10] and methods that separate point mutant from wild type sequences on the basis of lower thermal stability in wild type/mutant heteroduplexes as opposed to wild type homoduplexes [11][12][13][14][15][16][17][18][19] . A review of all of these contributions is beyond the scope of this effort; here we focus on the technical history and applications of methods that depend on the separation of wild type and mutant sequences on the basis of kinetics of DNA melting and reannealing while DNA is moved by electromotive force through a molecular sieve such as a polyacrylamide gel.…”

“…Fischer used a fixed temperature and a chemical gradient of urea/formamide to dramatically slow a homoduplex molecule as it approached the melting condition of the lower isomelting domain on a gel. Using this approach Fischer and Lerman separated homoduplex DNA sequences differing by but a single base pair 11,12 terming their technology, denaturing gradient gel electrophoresis or DGGE. Leonard Lerman kindly provided training for our laboratory in early 1984.…”