Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus.
A DNA fragment (307 bp) from the conserved region of an adenovirus gene (hexon) was amplified by symmetric and by asymmetric polymerase chain reaction (PCR). Two amplifications, one in the absence other in the presence of a molecular beacon probe were conducted by both symmetric and asymmetric PCR. The probe sequence was complementary to an internal segment of the amplified fragment. The product amplified in the absence and presence of the probe was detected by agarose gel and fluorescence analysis, respectively. A symmetric PCR results in exponentially grown double stranded DNA. An asymmetric PCR generates one of the strands by linear ampIlification and a fraction of its total product as double-stranded DNA limited by the concentration ratio of the primers used. Thus asymmetric PCR provided lower intensity signal hence less sensitivity than symmetric PCR by agarose gel analysis as expected. However, signal from a beacon probe based PCR assay is generated only from the probe fraction that hybridizes successfully competing against the strand complementary to the target strand of the product generated by PCR. The symmetric PCR has so far been used for the molecular beacon based fluorescent signal detection. The present study compared the level of fluorescent signal detectable from a symmetric PCR with that from an asymmetric PCR. The fluorescent data analysis demonstrated that a significant higher level of fluorescent signal hence higher sensitivity of detection is obtainable using asymmetric PCR than symmetric PCR performed in presence of the molecular beacon probe.
Poddar
S K
SK
Department of Pediatrics, Division of Infectious Diseases and Pediatric Pharmacology Research Unit (PPRU), La Jolla, CA 92093-0808, USA. poddar@nhrc.navy.mil
D000260:Adenoviruses, Human / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D005814:Genes, Viral / Q000235:genetics; D020319:Oligodeoxyribonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes; D020319:Oligodeoxyribonucleotides, Antisense