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Journal of Clinical Microbiology, May 2004, p. 1965-1976, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1965-1976.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

Matthew M. Ferris,1 Xiaomei Yan,1 Robbert C. Habbersett,1 Yulin Shou,1 Cheryl L. Lemanski,1 James H. Jett,1 Thomas M. Yoshida,2 and Babetta L. Marrone1*

Bioscience,1 Chemistry Divisions, Los Alamos National Laboratory, Los Alamos, New Mexico 875452

Received 6 October 2003/ Returned for modification 1 December 2003/ Accepted 27 January 2004

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 [RSDPFGE ] = 3% ± 2% and RSDFCM = 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.

* Corresponding author. Mailing address: Mail Stop M888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 667-3279. Fax: (505) 665-3024. E-mail: blm{at}lanl.gov.

Journal of Clinical Microbiology, May 2004, p. 1965-1976, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1965-1976.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zhong, W., Shou, Y., Yoshida, T. M., Marrone, B. L. (2007). Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis. Appl. Environ. Microbiol. 73: 3446-3449 [Abstract] [Full Text]  


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