Using the fluorogenic 5' nuclease assay for high-throughput detection of (CA)n repeats in radiation hybrid mapping.
Here, the power of the 5' nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5' nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using this strategy greatly enhanced the rapidity, throughput and accuracy of the RH mapping of microsatellite markers.
Jouquand
S
S
UPR 41 CNRS Recombinaisons génétiques, Faculté de Médecine, Rennes, France.
André
C
C
Chéron
A
A
Hitte
C
C
Chuat
J C
JC
Galibert
F
F
D015720:5'-Nucleotidase / Q000737:chemistry*; D000818:Animals; D018900:Dinucleotide Repeats / Q000235:genetics*; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006822:Hybrid Cells / Q000166:cytology / Q000528:radiation effects*; D018895:Microsatellite Repeats / Q000235:genetics; D020161:Physical Chromosome Mapping / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000737:chemistry
D005456:Fluorescent Dyes; D019914:Taq Polymerase; D015720:5'-Nucleotidase