Abstract
Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.
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- Cardullo R. A., Agrawal S., Flores C., Zamecnik P. C., Wolf D. E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clegg R. M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 1992;211:353–388. doi: 10.1016/0076-6879(92)11020-j. [DOI] [PubMed] [Google Scholar]
- Erlich H. A., Arnheim N. Genetic analysis using the polymerase chain reaction. Annu Rev Genet. 1992;26:479–506. doi: 10.1146/annurev.ge.26.120192.002403. [DOI] [PubMed] [Google Scholar]
- Holland P. M., Abramson R. D., Watson R., Gelfand D. H. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7276–7280. doi: 10.1073/pnas.88.16.7276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerem B., Rommens J. M., Buchanan J. A., Markiewicz D., Cox T. K., Chakravarti A., Buchwald M., Tsui L. C. Identification of the cystic fibrosis gene: genetic analysis. Science. 1989 Sep 8;245(4922):1073–1080. doi: 10.1126/science.2570460. [DOI] [PubMed] [Google Scholar]
- Matayoshi E. D., Wang G. T., Krafft G. A., Erickson J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science. 1990 Feb 23;247(4945):954–958. doi: 10.1126/science.2106161. [DOI] [PubMed] [Google Scholar]
- Morrison L. E., Halder T. C., Stols L. M. Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal Biochem. 1989 Dec;183(2):231–244. doi: 10.1016/0003-2697(89)90473-9. [DOI] [PubMed] [Google Scholar]