Fluorescence cross-correlation: A new concept for polymerase chain reaction

Rigler, Rudolf; Földes-Papp, Zeno; Meyer‐Almes, Franz‐Josef; Sammet, Cyra; Völcker, Martin; Schnetz, Andreas

doi:10.1016/s0168-1656(98)00079-0

Cited by 122 publications

(116 citation statements)

References 22 publications

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“…A large central peak is observed, resulting from the coincident detection of red and green fluorescent bursts, due to the large number of PCR amplicons incorporating both primers. Histograms for each PCR cycle analyzed in addition to cycles 0 and 40 ͑cycles 4,8,12,16,20,24,28, 32, results not shown͒ display an increasing number of amplicons detected as they are synthesized over the course of the reaction. Around 15,000 photon bursts of each color were detected in 15 sequential one minute runs for each cycle number analyzed.…”

Section: Photon Burst Analysis and Single Molecule Detectionmentioning

confidence: 94%

“…Both primers were added at a final concentration of 0.2 M. Genomic DNA ͑1 l per reaction, approximately 50 ng of DNA͒ was added and 50 l reactions were performed with an Icycler from Bio-Rad ͑Hercules, CA͒ using the following amplification program: 94°C for 5 min for initial denaturation, then 94°C for 30 s, 55°C for 20 s, 72°C for 20 s, and 72°C for 5 min for the final extension. Tubes were sequentially removed and chilled on ice after 0, 4,8,12,14,16,20,22,24,26,28,30,32,34,36,38, and 40 cycles. PCR samples were diluted to approximately 100 pM in 5X TBE with 1% ͑wt/ vol͒ poly͑vinylpyrrolidone͒ added to suppress electroosmotic flow 29 and surface interactions for analysis in submicrometer fluidic channels.…”

Section: A Samplesmentioning

confidence: 99%

“…8,[16][17][18] In the late 1990s, dual-color FCCS was established as a viable experimental method, 27 and was later used to quantify a PCR reaction in free solution. 28 In order to determine analyte flow speed using FCS and FCCS, the fluorescence intensity of the two detection channels were both autocorrelated and cross-correlated. The resulting correlation functions were fit to analytical models to extract information pertaining to the flow speeds of the various analytes.…”

Section: Introductionmentioning

confidence: 99%

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Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

et al. 2007

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Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction ͑PCR͒ products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca ͑325 bp͒ were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and crosscorrelation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity.

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Section: Photon Burst Analysis and Single Molecule Detectionmentioning

confidence: 94%

Section: A Samplesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

et al. 2007

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“…The purpose of this study was to investigate the mechanism of exogenous DNA degradation in situ, in cytoplasm, by analyses of diffusion properties of DNAs and to determine the effects of cytoplasmic degradation on the gene expression rate. Thus we employed FCS [10,[16][17][18] to measure the diffusion properties of DNAs and FCCS [18][19][20][21][22] to monitor the degradation of DNAs by cytoplasmic nucleases at the single molecule level. Furthermore, we predicted that exonucleases would work as the main barrier in cytoplasm, so a capped structure was attached to the transfected DNA to enhance the expression of the protein EGFP.…”

Section: Introductionmentioning

confidence: 99%

Monitoring intracellular degradation of exogenous DNA using diffusion properties

Sasaki¹,

Kinjo²

2010

Journal of Controlled Release

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Artificial nonviral gene vectors have the potential to improve the safety of gene therapy; however, such artificial vectors are less efficient for gene expression due to the existence of various barriers to delivery of exogenous DNAs to the nucleus in the living cell. Here we describe the degradation activities of cytoplasmic nucleases, which are involved as a barrier to efficient exogenous gene expression. The size and structure of degraded DNA were monitored by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) in solution and in the cytoplasm of living cells. Differences in degradation by endo-and exonucleases were confirmed by differences in the size and structure of DNA. Moreover, we confirmed the influence of the exonuclease degradation in cytoplasm on the expression rate of DNA transfection with a cationic lipid. Based on a comparison of in vitro and cell measurements, we conclude that cytoplasmic degradation by exonucleases can be a considerable barrier against efficient gene delivery.

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“…In the last few years an increasing number of applications of dual-colour FCCS have been reported. These vary from enzyme kinetics Koltermann et al 1998, Rarbach et al 2001, nucleotide hybridisation (Schwille et al 1997;Rigler et al 1999b) and protein-DNA interactions (Rippe 2000) to the application of two-photon excitation (Heinze et al 2000). Dual-colour FCCS applications in live cells have been reported recently (Bacia et al 2002;Hink et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Global analysis of fluorescence fluctuation data

et al. 2005

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Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.

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Fluorescence cross-correlation: A new concept for polymerase chain reaction

Cited by 122 publications

References 22 publications

Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Monitoring intracellular degradation of exogenous DNA using diffusion properties

Global analysis of fluorescence fluctuation data

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