Symmetric vs asymmetric PCR and molecular beacon probe in the detection ofa target gene of adenovirus

A DNA fragment (307 bp) from the conserved region of an adenovirus gene (he xon) was amplified by symmetric and by asymmetric polymerase chain reaction (PCR). Two amplifications, one in the absence other in the presence of a m olecular beacon probe were conducted by both symmetric and asymmetric PCR. The probe sequence was complementary to an internal segment of the amplifie d fragment. The product amplified in the absence and presence of the probe was detected by agarose gel and fluorescence analysis, respectively. A symm etric PCR results in exponentially grown double stranded DNA. An asymmetric PCR generates one of the strands by linear amplification and a fraction of its total product as double-stranded DNA limited by the concentration rati o of the primers used. Thus asymmetric PCR provided lower intensity signal hence less sensitivity than symmetric PCR by agarose gel analysis as expect ed. However, signal from a beacon probe based PCR assay is generated only f rom the probe fraction that hybridizes successfully competing against the s trand complementary to the target strand of the product generated by PCR. T he symmetric PCR has so far been used for the molecular beacon based fluore scent signal detection. The present study compared the level of fluorescent signal detectable from a symmetric PCR with that from an asymmetric PCR. T he fluorescent data analysis demonstrated that a significant higher level o f fluorescent signal hence higher sensitivity of detection is obtainable us ing asymmetric PCR than symmetric PCR performed in presence of the molecula r beacon probe. (C) 2000 Academic Press.