Aberrant DNA sequence pattern caused by a heterozygosity.

The polymerase chain reaction (PCR) and DNA sequence determination (4,5) represent two of the most routine and reliable approaches to determine genic sequence variation. Several automated and manual sequencing kits are commercially available, and they provide adequate and satisfactory results. Compressions caused by formation of secondary structure, however, remain particularly problematic when performing manual sequencing using radiolabeled primers. Use of 7deaza-dGTP instead of dGTP can improve sequence quality and reduce compression (8), but mutations can also cause anomalous sequence patterns (9,10) that could lead to false interpretation of data. A polymorphism in the human uncoupling protein 3 (UCP3) gene in African-Americans was recently identified in our laboratory (1). This polymorphism was defined by a G→A transition at position 304 of the cDNA (c.304G/A). DNA sequence determination of the polymorphism-containing region with the reverse primer showed a complex electrophoretic pattern present only in heterozygotes. This aberrant sequence pattern was eliminated when heterozygotes were sequenced with the forward primer, suggesting that upstream sequencing and heterozygosity could cause electrophoretic artifacts. DNA amplification. Total genomic DNA (50–100 ng) was used to amplify the third coding exon of the UCP3 gene, with the following primers: [forward primer: UCP3e3f: 5′-TGACCAGCATGGTTGTTCTA-3′; reverse primer: UCP3e3r: 5′-CCTGGTCTGCCTCTGAGTCT-3′]. Cycling conditions and purification of the 376bp PCR product for sequencing were performed as previously described (1). DNA sequence determination. Purified PCR products were directly sequenced with the AmpliCycle Sequencing Kit as recommended by the manufacturer (PE Biosystems, Foster City, CA, USA), using an end-labeled (ATP, γ-33P) primer (UCP3e3f or UCP3e3r). Additional sequencing was performed with the Sequenase Sequencing Kit and the Thermo Sequenase Sequencing Kit (both from Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to manufacturer’s instructions. Sequence determination was performed manually in casted gels using the Gel-Mix 6-Sequencing Gel Solution (Life Technologies, Gaithers-