BIPOLAR CLAMPING IMPROVES THE SENSITIVITY OF MUTATION DETECTION BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

Temperature gradient gel electrophoresis (TGGE) is a rapid and sensiti ve screening method for point mutations and other small DNA alteration s. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a ''GC-clam p'' is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mu tation within the fragment is detected through the presence of three a dditional bands in the TGGE gel, the mutant homoduplex and two heterod uplex bands. However, the ideal pattern of four sharp bands is not alw ays found due to inconsistencies in melting behavior along the sequenc e of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Her e, we describe a method to overcome this problem by utilizing one psor alen clamp at each end of the PCR product. Using TGGE assays establish ed for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN 1 gene as examples, we show that bipolar clamping may transform blurre d bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.