No abstract

Melting of DNA in a segment bounded at both ends by regions of greater stability during electphoresis in denaturing gradient gels shows complex properties, not accommodated within standard meiting theory. Compact bands of some DNA molecules become anomalously broadened at the retardation level in a denaturing gradient, or double bands may appear in a uniform denaturant concentration. These properties are associated only with molecules for which the distribution of stability calculated by the Poland-Fixman-Freire algorithms indicates that the region of lowest stability does not extend to an end of the molecule. Retention of helicity at the ends is shown by the difference in the effect of base substitution in the end domains and in the least stable domain. Both the appearance of double bands and band broadening can be explained by invoking a hypothetical metastable intermediate in meiting, which is converted into the equilibrium melted form at a relatively slow rate, depending on both denaturant concentration and field strength. A kinetic model permits plausible rate constants to be inferred from the pattems. Despite the increased band width, sequence variants with base changes in the least stable domain result in readily detectable band shifts in the gradient. INTRODUCTIONThe stepwise melting of DNA molecules of length >100 bp in a gradually increasing denaturing environment is attributable to strong cooperativity and suppression of the unraveling of short interior loops or bubbles. The steps correspond to the concurrent melting within a narrow temperature interval of a large number of contiguous bases, called domains. Denaturing gradient gel electrophoresis (DGGE) is sufficiently sensitive to detect and measure the change in melting temperature of a domain resulting from substitution of a single base. It provides a general means for detection of gene mutations and polymorphisms.Electrophoresis in a denaturing gradient differs from conventional gel electrophoresis in that the mobility of a fragment is not constant,

T o search for mutations in the human KRAS2 oncogene, we have analyzed polymerase chain reaction (PCR) fragments by denaturing gradient gel electrophoresis. W e used six different PCR fragments to screen the five coding exons of this gene as well as the splice sites. G C clamps were added to each fragment by means of heteroduplex extension. W e infer, from a theoretical analysis in which we employed the computer programs MELT and SQHTX, that virtually any mutation affecting one o r a few base pairs in the coding exons or splice sites will be detectable. Thus the system that we describe should allow comprehensive detection of mutations throughout the coding exons and splice sites of the KRAS2 gene. As an example, we show that missense mutations at codons 12, 13, and 6 I can be detected; mutant KRAS2 genes isolated from human tumors have been found t o contain mutations only at these codons. W e also report the discovery of three new polymorphic loci in the KRASZ gene. W e show that IPCR reactions containing two o r three fragments can be screened in a single lane of a denaturing gradient gel; this dramatically increases the number of base pairs that can be screened per gel. Genes Chrom Cancer 6:73-85 (1993).