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 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...

Analysis of Listeria monocytogenes Strain Distribution in a Pork Slaughter and Cutting Plant in the Province of Quebec

“…Similarity between the patterns was determined based on band positions by using the Dice correlation with a position tolerance of 1% (18). The strains were clustered using the unweighted pair group method with arithmetic means, and a dendrogram was then constructed (18). For each different combined Apal and A id PFGE profile highlighted, a pulsotype number was attributed.…”

“…Patterns were compared using BioNumerics software (version 6.5, Applied Maths, Kortrijk, Belgium). Similarity between the patterns was determined based on band positions by using the Dice correlation with a position tolerance of 1% (18). The strains were clustered using the unweighted pair group method with arithmetic means, and a dendrogram was then constructed (18).…”

Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

“…729 The requisite throughput is thus quite small; in principle, a fingerprint containing 40 restriction fragments could be sized in less than a minute. In practice the analysis time is somewhat longer (e.g., 30 minutes 100 ), which is still much faster than pulsed field gel electrophoresis if one skips the electroelution step 723 and instead dissolves the gel using GELase. 100 It is possible to increase the throughput of the measurement to 2000 fragments/s by using a planar sheet of laser light and imaging the DNA passing through the laser sheet using a CCD camera.…”

“…In practice the analysis time is somewhat longer (e.g., 30 minutes 100 ), which is still much faster than pulsed field gel electrophoresis if one skips the electroelution step 723 and instead dissolves the gel using GELase. 100 It is possible to increase the throughput of the measurement to 2000 fragments/s by using a planar sheet of laser light and imaging the DNA passing through the laser sheet using a CCD camera. 728 While it is certainly desirable to increase the throughput of the measurement, the CCD imaging method is prone to numerous artifacts due to the limited readout time from the camera and the different flow velocities at different points inside the illuminated region.…”

Analysis of single nucleic acid molecules in micro- and nano-fluidics

“…When using this method sizing resolution is limited by two factors: statistical variation in the ratio of dyes per nucleotide between DNA molecules, and detector sensitivity and uniformity over the excitation volume. The staining ratio inherently limits sizing resolution and makes the method better suited to sizing of larger DNA molecules (kilobasepairs), although sizing of 125 bp fragments has been demonstrated 52 . The other factor affecting burst size variation is the uniformity of excitation and collection throughout the detection volume.…”