DOI: 10.1073/pnas.93.23.12805. Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

10.1073/pnas.93.23.12805

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Bibliography

Walter, N. G., Schwille, P., & Eigen, M. (1996). Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection byâPCR. Proceedings of the National Academy of Sciences, 93(23), 12805â12810.

Authors 3

Nils G. Walter (first)
Petra Schwille (additional)
Manfred Eigen (additional)

References 38 Referenced 44

10.1103/PhysRevLett.29.705
10.1002/bip.1974.360130102
M Ehrenberg, R Rigler J Chem Phys 4, 390–401 (1974). / J Chem Phys by Ehrenberg M (1974)
N L Thompson Topics in Fluorescence Spectroscopy, ed J R Lakowicz (Plenum, New York) 1, 337–378 (1991). / Topics in Fluorescence Spectroscopy by Thompson N L (1991)
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10.1016/0005-2787(80)90053-2
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A Schober, N G W Walter, U Tangen, G Strunk, T Ederhof, J Dapprich, M Eigen BioTechniques 18, 652–660 (1995). / BioTechniques by Schober A (1995)
10.1073/pnas.93.23.12811
10.1093/nar/20.7.1691
10.1016/0168-1656(95)00054-T

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:43 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 3:21 p.m.)
Indexed	1 year, 10 months ago (Oct. 18, 2023, 4:53 a.m.)
Issued	28 years, 9 months ago (Nov. 12, 1996)
Published	28 years, 9 months ago (Nov. 12, 1996)
Published Online	28 years, 9 months ago (Nov. 12, 1996)
Published Print	28 years, 9 months ago (Nov. 12, 1996)

Funders 0

None

BibTeX

@article{Walter_1996, title={Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR}, volume={93}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.93.23.12805}, DOI={10.1073/pnas.93.23.12805}, number={23}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Walter, Nils G. and Schwille, Petra and Eigen, Manfred}, year={1996}, month=nov, pages={12805–12810} }

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  "abstract": "<jats:p>\n            A sensitive, labor-saving, and easily automatable\n nonradioactive procedure named APEX-FCS (amplified probe extension\n detected by fluorescence correlation spectroscopy) has been established\n to detect specific\n            <jats:italic>in vitro</jats:italic>\n            amplification of pathogen\n genomic sequences. As an example,\n            <jats:italic>Mycobacterium\n tuberculosis</jats:italic>\n            genomic DNA was subjected to PCR amplification\n with the Stoffel fragment of\n            <jats:italic>Thermus aquaticus</jats:italic>\n            DNA\n polymerase in the presence of nanomolar concentrations of a\n rhodamine-labeled probe (third primer), binding to the target in\n between the micromolar amplification primers. The probe becomes\n extended only when specific amplification occurs. Its low concentration\n avoids false-positives due to unspecific hybridization under PCR\n conditions. With increasing portion of extended probe molecules, the\n probe\u2019s average translational diffusion properties gradually change\n over the course of the reaction, reflecting amplification kinetics.\n Following PCR, this change from a stage of high to a stage of low\n mobility can directly be monitored during a 30-s measurement using a\n fluorescence correlation spectroscopy device. Quantitation down to 10\n target molecules in a background of 2.5 \u03bcg unspecific DNA without\n post-PCR probe manipulations could be achieved with different\n primer/probe combinations. The assay holds the promise to\n concurrently perform amplification, probe hybridization, and specific\n detection without opening the reaction chamber, if sealable foils are\n used.\n          </jats:p>",
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