The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ؎ 2%) and FCM (4% ؎ 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD PFGE ] ‫؍‬ 3% ؎ 2% and RSD FCM ‫؍‬ 1.2% ؎ 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional bandbased methods.DNA fragment sizing is arguably the most widely used analytical method in molecular biology, biochemistry, and microbiology. Specific applications of DNA fragment sizing include microbe identification and discrimination, genotyping, and sequencing. Traditionally, DNA fragments are characterized via size-dependent separation methods such as gel electrophoresis.Conventional gel electrophoresis, using a solid polymer (e.g., agarose or polyacrylamide) and a static electric field, is ubiquitous in today's molecular biology and biochemistry labs. Such methods are routinely used to separate and size DNA fragments of Յ20 kb (4).Pulsed-field gel electrophoresis (PFGE), developed in 1984 by Schwartz et al. (44), extended the fragment sizing range to over 1 Mb (4,6,9,19,44). For PFGE, large DNA fragments (i.e., Ͼ10 kb) are separated in an agarose gel by a pulsed electric field. A popular application of PFGE has been strainlevel bacterial fingerprinting through the sizing of DNA fragments resulting from the digestion of whole genomic DNAs with rare-cutting restriction endonucleases (5,11,28,35,47,49). Macrorestriction-based bacterial fingerprinting has found applications primarily in the public health and food safety industries. For example, the Centers for Disease Control and Prevention (CDC) have formed PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance (46; http://www.cdc.gov/pulsenet). PulseNet utilizes macrorestriction fi...

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 Ͼ 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 singlemolecule 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