Reverse transcription-polymerase chain reaction (RT-PCR) is frequently used to simultaneously detect mRNA isoforms, which are generated by alternative splicing. Here we characterize two previously unrecognized RT-PCR products of vascular endothelial growth factor (VEGF) RNA. DNA products with apparent sizes of 600 and 1200 base pairs (bp) were detected at high cycle numbers. Heat denaturation of the smaller product and subsequent reannealing revealed that it was a heteroduplex consisting of two

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... different DNA strands. These were identified by DNA sequencing as the amplification products of two VEGF transcripts, i.e. VEGF 121 and VEGF 165 , which differ by the presence of one exon. S1 nuclease analysis showed that this exon is bulged out as a single-stranded loop. Purified heteroduplexes in solution were found to form a 1200-bp DNA product which could be reconverted into 600-bp DNA heteroduplexes by mild denaturation at 70°C. These findings suggest that this product is formed by base pairing of complementary heteroduplex loops and represents a novel four-stranded DNA structure.