Double-labeled fluorescent probes for 5' nuclease assays: purification and performance evaluation

Biotechniques. 1997 Jun;22(6):1140-5. 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 Taq 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 5'-Nucleotidase / analysis*
  • Adenosine Triphosphate / metabolism
  • Chromatography, High Pressure Liquid
  • DNA Primers
  • DNA Probes, HLA*
  • DNA-Directed DNA Polymerase / metabolism
  • Fluorescent Dyes / isolation & purification
  • Fluorescent Dyes / metabolism*
  • Fluorometry
  • HLA-DR Antigens / genetics
  • Humans
  • Nucleic Acid Conformation
  • Oligonucleotide Probes*
  • Polymerase Chain Reaction / methods*
  • Taq Polymerase


  • DNA Primers
  • DNA Probes, HLA
  • Fluorescent Dyes
  • HLA-DR Antigens
  • Oligonucleotide Probes
  • Adenosine Triphosphate
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • 5'-Nucleotidase