No abstract

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguih two DNA molecules that differ by as little as a single-base substitution. This method detects =50% of all possible single-base changes in DNA fragments ranging from 50 to -1000 base pairs. To increase the number of single-base changes that can be distingished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G+C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human 13-globin genes. We show that this GC-clamp allows the detection of mutations, includig the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human P-globin gene, that were previously indiishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from <5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.Genetic linkage analysis has been severely limited in humans due to a paucity of informative polymorphic loci. The recognition that DNA sequence polymorphism can fill this void has revolutionized the field of human genetics (1, 2). Consequently, approaches that allow the detection of singlebase differences in specific regions of genomic DNA have been extremely powerful for both linkage analysis and direct detection of mutations associated with human disease (3-9). The vast majority of base changes have been identified by the restriction fragment length polymorphism approach, which measures DNA sequence alterations due to a loss or gain of a restriction enzyme cleavage site or to variation in length caused by deletion or insertion (1). However, many singlebase changes do not alter a restriction enzyme cleavage site and, therefore, cannot be detected by the restriction fragment length polymorphism method. One alternative method that makes it possible to detect a larger fraction of all possible base changes in a DNA fragment is denaturing gradient gel electrophoresis (DGGE;.DGGE is a gel system that separates DNA fragments according to their melting properties (10-12). When a DNA fragment is electrophoresed through a linearly increasing gradient of denaturants, the fragment remains double stranded until it reaches the concentration of denaturants equivalent to a melting temperature (tm) that causes the lower-temperature melting domains of the fragment to melt. At this point, the branching of the molecule caused by partial melting sharply decreases the mobility of the fragment in the gel. The lower-temperature melting domains of DNA fragments differing by as little as a single-base substitution will melt at slightly different denaturant concentrations because ofdifferences in stacking interactions between adja...