Double-Labeled Fluorescent Probes for 5′ Nuclease Assays: Purification and Performance Evaluation

• Published in: BioTechniques Vol. 22; no. 6; pp. 1140 - 1145
• Main Authors: Rudert, W.A, Braun, E.R, Faas, S.J, Menon, R, Jaquins-Gerstl, A, Trucco, M
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.06.1997; Eaton
• Subjects: 5'-Nucleotidase - analysis; Adenosine Triphosphate - metabolism; Biological and medical sciences; Biotechnology; Chromatography, High Pressure Liquid; Diverse techniques; DNA Primers; DNA Probes, HLA; DNA-Directed DNA Polymerase - metabolism; Fluorescent Dyes - isolation & purification; Fluorescent Dyes - metabolism; Fluorometry; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; HLA-DR Antigens - genetics; Humans; Methods. Procedures. Technologies; Miscellaneous; Molecular and cellular biology; Nucleic Acid Conformation; Oligonucleotide Probes; Polymerase Chain Reaction - methods; Synthetic digonucleotides and genes. Sequencing; Taq Polymerase; Performance evaluation; Polymerase chain reaction; Purification; Fluorescent labelling; HPLC chromatography; Molecular probe; Method; Application
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/97226rr02

An inexpensive method for the purification and evaluation of user-synthesized or crude commercially prepared double-labeled fluorescent probes is presented. These probes exhibit the characteristics required for use in 5′-nuclease assays, including efficient reporter dye quenching, target specificity and susceptibility to cleavage by DNA polymerase during PCR amplification. The method is suitable for research laboratories that wish to develop 5′ nuclease assays for the detection of PCR-amplified target sequences to eliminate the requirement for agarose gels and to advance throughput.