The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice

H. J. Hnatyszyn, E. R. Podack, A. K. Young, R. Seivright, G. Spruill, G. Kraus

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.

Original languageEnglish (US)
Pages (from-to)169-175
Number of pages7
JournalMolecular and Cellular Probes
Volume15
Issue number3
DOIs
StatePublished - Jan 1 2001

Keywords

  • Fluorogenic probes
  • FRET
  • Genotype
  • gld
  • Mutation
  • Real-time PCR

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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