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Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations

se from the PCR/RFLP analysis. Third, 154 genotype-unknown human genomic DNA samples were genotyped by molecular beacon analysis for the three mutations, and results were confirmed for selected samples by DNA sequencing.

Conclusions

Use Stratagenes single-tube allelic discrimination assays to realize several advantages over existing mutation detection techniques: The hairpin-shaped molecular beacon probes are more specific in distinguishing single base-pair mismatches than linear probes.2 Because the test is performed in a closed tube and no post-PCR manipulation of samples is required, the test saves time and effort and significantly reduces the risk of PCR product carry-over contamination. Being able to use two allele-specific molecular beacons in the same PCR solution enables three possible genotype representations (two homozygotes and a heterozygote) of two allelic variants in target DNA to be determined at the same time. It also definitively discriminates a true negative result from a false negative result due to PCR failure. Finally, the technology can be adapted for high-throughput assays. Currently, with the 96-well PCR plate, it takes about 4 hours to completely screen 96 DNA samples; further enhancement of the assay throughput can be achieved by using a higher density plate format (e.g., 384-well format).

Acknowledgments

The authors thank the following: Dr. P.N. Ray (Molecular Diagnostic Laboratory, the Hospital for Sick Children, Toronto, Canada) for providing the CF genotype-specific human genomic DNA; Dr. J.W. Longshore (Molecular Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, SC) and Dr. V.M. Pratt (LabCorp, Research Triangle Park, NC) for providing the factor II genotype-specific human genomic DNA; Dr. J.W. Longshore (Molecular Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, SC) and Dr. C. Mura (Laboratoi
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