DNA diagnostic has been moving from expensive, low-throughput, multistep methods to inexpensive, higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays. In this method, we describe a universal molecular beacon (U-MB) as the fluorescent tracer in the real-time PCR technique. A 5'-universal template primer (5'-UT primer) has been designed with a tail in complementary to the loop and 5'-side arm sequence of U-MB at the 5'-end of forward target specific primer. As PCR cycles increase, a new DNA fragment with a 5'-UT primer tail is synthesized, which is used as the template for next PCR cycle. As the reverse primer extends to the 5'-UT primer tail, the U-MB hybridized is displaced and the fluorescence from the fluorophore of the U-MB is quenched, indicating that the allele-specific PCR is in progress. This tracing system combined with an allele-specific reverse primer and vent (exo-) DNA polymerase, a polymerase that lacks 3'- to 5'-exonuclease activity, was used for the detection of point mutations of base G in codon 259 (AGA) of exon 7 of p53 gene on a panel of breast cancer individuals.
A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer-dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method.
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