Confocal data acquisition for digital quantification using amplified single molecule detection

Jonas Melin, Jonas Jarvius, Jenny Göransson, Johan Stenberg, Fredrik Nikolajeff, Mats Nilsson*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingConference proceeding contributionpeer-review

Abstract

We have recently presented a method that enables single molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible by counting the number of generated RCPs using standard epi-fluorescence or confocal fluorescence microscopes. We have characterized the performance of the epi-fluorescence and the confocal readout formats. Both formats exhibit a linear response of the number of counted objects as a function of starting circles, and the dynamic range is three orders of magnitude employing epi-fluorescence readout and four when using confocal. In the epi-fluorescence format flow rate has to be below 1 μ1/min and flow variations are likely to be the limiting factor for precision. If the flow rate is above 3 μ1/min the precision of the confocal readout format is limited only by Poisson counting statistics, due to the accurate volume definition of the confocal optics. The limit of detection in the confocal format was reduced by a factor of three by increasing the data acquisition rate by a factor of ten.

Original languageEnglish
Title of host publicationOptically Based Biological and Chemical Detection for Defence III
Volume6398
DOIs
Publication statusPublished - 2006
EventOptically Based Biological and Chemical Detection for Defence III - Stockholm, Sweden
Duration: 11 Sep 200613 Sep 2006

Other

OtherOptically Based Biological and Chemical Detection for Defence III
CountrySweden
CityStockholm
Period11/09/0613/09/06

Keywords

  • Confocal detection
  • DNA
  • Padlock probes
  • Single molecule detection

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