Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis.
Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. 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-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.
Gille
C
C
Institut für Medizinische Genetik, Universitätsklinikum Charité, Berlin, Germany.
Gille
A
A
Booms
P
P
Robinson
P N
PN
Nürnberg
P
P
D004247:DNA / Q000032:analysis*; D004586:Electrophoresis / Q000379:methods*; D000071837:Fibrillins; D008840:Microfilament Proteins / Q000235:genetics; D009154:Mutation; D025542:Neurofibromin 1; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature
D000071837:Fibrillins; D008840:Microfilament Proteins; D025542:Neurofibromin 1; D011506:Proteins; D004247:DNA