Universal molecular beacon-based tracer system for real-time polymerase chain reaction.
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.
Li
Xiaomin
X
The Key Laboratory of Bioorganic Chemistry & Molecular Engineering and Institute of Analytical Chemistry, College of Chemistry & Molecular Engineering, Peking University, Beijing, 100871, China.
Huang
Yong
Y
Guan
Yuan
Y
Zhao
Meiping
M
Li
Yuanzong
Y
D001943:Breast Neoplasms / Q000235:genetics; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005260:Female; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D013698:Templates, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology
D003062:Codon; D017931:DNA Primers; D015345:Oligonucleotide Probes; C034477:RNA primers; D016159:Tumor Suppressor Protein p53; D012313:RNA; D012321:DNA-Directed RNA Polymerases