Use of chemical clamps in denaturing gradient gel electrophoresis: application in the detection of the most frequent Mediterranean beta-thalassemic mutations.

Institut National de la Sant~ et de la Recherche M~dicale (INSERM) U 129, Institut Cochin de Genetique Moleculaire (ICGM), 75014, Paris Cedex, France The most frequent molecular defects in genetic diseases are represented by point mutations. There is an increasing need for practical, efficient, and inexpensive ways to explore mutations responsible for these diseases. PCR has solved the problem of the target. When the precise site of a point mutation is not known, it is necessary to first determine the region harboring the defect. Six methods are currently available: single-strand conformation polymorphisms (SSCP), chemical cleavage, RNase mismatch cleavage, reaction of DNA heteroduplexes with a water-soluble carbodiimide, direct sequencing, and denaturing gradient gel electrophoresis (DGGE). To be used routinely, a method must be safe, simple, and of reasonable cost. The first five methods involve unsafe reagents (chemicals and radioactivity), but in some cases are relatively inexpensive. The last method, DGGE, is safe but very expensive, because of the high cost of the long GC sequence (from 30 to 80 nucleotides) that must be attached to the 5' end of one of the primers. In addition, with such a clamp, the yield of primer is reduced drastically, increasing the cost of experiments. Chemical clamps (AppligEne, Strasbourg, France) have been designed recently, allowing these difficulties to be overcome. Instead of adding a long GC-rich tail, the end of the DNA fragment can be stabilized by covalently linking both strands with a chemical agent. The best such agent currently available is psoralen (furocoumarin), (1) a photoactivatable intercalating agent. When irradiated, this intercalated polycyclic compound becomes covalently linked to the 5-6 double bonds of the complementary strands (particularly thymine). The substance is chemically fixed by the manufacturer (AppligEne) to the 5' end (including two adenines) of the primer to be clamped. This procedure represents a very attractive alternative to GC clamping, especially as most laboratories in the world do not have an oligonucleotide synthesizer. To extend this method, we tested it on known mutations responsible for [3-thalassemia in the Mediterranean region. This disease is the most common recessive inherited disorder in Mediterranean populations, (2~ and in the last few years the molecular basis of [3-thalassemia in this area, as well as in other populations, has been largely defined by cloning and sequencing or oligonucleotide analysis. More than 110 different mutations, heterogeneously distributed, have been reported to cause this disease. (3,4~ Here, we propose the use of chemical clamps in combination with the DGGE method for a single-assay diagnosis of the most frequent [3-thalassemic mutations encountered in Mediterranean countries. (s~