Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis

Abstract

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 band-based methods.

FIG. 1.

Comparison of PFGE and FCM RFLP fingerprints of SmaI-digested S. aureus Mu50. Representative data sets for PFGE and FCM are shown, with each data set represented as both a densitometric curve (or histogram) and a gel (or histogel) lane. Additionally, a histogram depicting the virtual digest fragment sizes (≥3.104 kb) is shown for reference. The PFGE and FCM peaks and bands at approximately 23, 58, and 110 kb are attributed to unresolved RFLP fragments (i.e., doublets), while the remaining peaks have been assigned to single RFLP fragments. Histograms have been vertically offset and normalized to allow for simultaneous viewing. The aspect ratios of the gel lane images were modified to match the histogram distributions and visually aligned with the curves for demonstration purposes.

FIG. 2.

FCM-generated fragment size histograms corresponding to the SmaI-digested Mu50 sample L3D1R2. The histogram used for calibration (a) contains three internal standards (17.4, 48.5, and 165.6 kb) in addition to the bacterial restriction fragments. Peaks corresponding to the internal standards were fit with a sum of three Gaussian peaks, and the peak centroid values (triangles) were used to create the calibration curve shown in the overlay. The linear regression fit to this curve, shown as a dashed line, allowed the raw FCM histogram to be calibrated in kilobase units and was obtained with a correlation coefficient (R) of 0.999. The calibrated fragment size scale was transferred to the histogram of the sample without internal standards added (b) after alignment of the two histograms was optimized by cross-correlation.

FIG. 3.

Band-based PFGE dendrogram for 45 replicates of SmaI-digested Mu50 and two clinical S. aureus isolates. The dendrogram shows the corresponding gel lane image for each sample beside its key, which indicates the experimental parameters. The error flags at each node indicate ±1 σ from the average similarity, and cophenetic coefficients are provided, in parentheses, for important nodes.

FIG. 4.

Band-based FCM dendrogram for 45 replicates of SmaI-digested Mu50 and two clinical S. aureus isolates.

FIG. 5.

Curve-based PFGE dendrogram for 45 replicates of SmaI-digested Mu50 and two clinical S. aureus isolates.

FIG. 6.

Curve-based FCM dendrogram for 45 replicates of SmaI-digested Mu50 and two clinical S. aureus isolates.