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Updated: Aug 23, 2026
A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA
Published on: October 1, 2017
Statistics of single-molecule measurements: applications in flow-cytometry sizing of DNA fragments
Matthew M Ferris1, Robbert C Habbersett, Murray Wolinsky
1Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Background:
The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters.
Methods:
Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of single-molecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM).
Results:
Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five single-molecule measurements would provide a mean value within one SD of the population mean. Additional simulations determined the number of measurements required to obtain the desired number of replicates for each subpopulation within a mixture. Application of these results to flow cytometry data for lambda/HindIII and S. aureus Mu50/SmaI DNA digests produced accurate DNA fingerprints from as few as 98 single-molecule measurements.
Conclusions:
A surprisingly small number of single-molecule measurements are required to obtain a mean measurement descriptive of a normally-distributed parent population.
