# Presentation: 722394

## Bacterial Strain Analysis of RFLP using DNA Fragment Sizing Flow Cytometry Cheryl L. Lemanski, Xiaomei Yan, Robert C. Habbersett, Thomas M. Yoshida, James H. Jett, Babetta L. Marrone Biosciences Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545

**Introduction:**** Restriction fragment length polymorphism (RFLP) followed by Pulsed-field gel electrophoresis (PFGE) has been the gold standard for bacterial strain identification and epidemiological analysis in public health settings to date. Although the specificity and reliability of RFLP/PFGE has made it a very powerful tool, PFGE has several deficiencies: it requires 20+ hours, micrograms of DNA, plus it is highly non-linear, and lacks direct quantitation.  We have developed an alternative technique, which is 100-fold faster and 10,000 fold more sensitive than the PFGE method.  In our approach, the DNA fragments, produced by digestion of the bacterial genome with a rare-cutting restriction endonuclease, are extracted from the agarose plug by GELase treatment, stained with a DNA intercalating dye (e.g. SYTOX-Orange or PicoGreen), and analyzed on a laboratory-built high sensitivity flow cytometer. **

**Acknowledgements**
**This work was supported by the Chemical and Biological Nonproliferation Program (NN-20) of the Department of Energy, by the NIH-funded National Flow Cytometry Resource (PR-01315), and by the FBI Hazardous Materials Response Unit, for which we are most grateful.  The statements and conclusions herein are those of the authors and do not necessarily represent the views of the FBI. Los Alamos Unclassified Report #02-7731.  **

**Stained bacterial/RFLP digest running through the high sensitivity flow cytometer individually, ****generating a fluorescence burst that is proportional to the size of the fragment. A histogram of the fragment sizes is generated and this "fingerprint" is comparable to the PFGE fingerprint used for bacterial strain identification.**

**Conclusions:**
**Our unique HSFCM offers more advantages over traditional PEGE for bacterial RFLP fingerprinting at the strain level.  Various bacterial genus, species, and strains have been successfully analyzed by DNA fragment sizing flow cytometry. **
** ****Linear relationship between DNA fragment length and burst fluorescence,**
** ****Accurate measurement of DNA fragment length from 125 bp to 400 kbp,**
** ****Fast analysis speed for fingerprint generation (10 min versus 20 hours),**
** ****High sensitivity (10 pg DNA versus ug),**
** ****Better resolution,**
** ****Excellent reproducibility.  **

**Laboratory-built compact high sensitivity flow cytometer (HSFCM)**

**Rapid DNA preparation for RFLP-based bacterial fingerprinting**

**Large DNA fragments recovered intact upon GELase procedure**

**Burst size distribution histogram of 1 kbp DNA stepladder**

**Accurate DNA fragment sizing by internal standardization **

**Our calibration procedure uses internal staining standards added directly to a sample of unknown fragments.  The standards, which must be recognizable against the peaks from unknown fragments in the sample, serve as signposts for linear regression analysis of the observed peak positions (of the standards) against their known sizes.  Peak positions are objectively determined by fitting a sum of Gaussians and a background curve to the peaks from the standards.  The derived slope and intercept are applied to the sample with staining standards, which calibrates the X-axis scale of the histogram.  In the second step, the histogram from the corresponding sample - without standards - is then superimposed (on the calibrated histogram), and the slope and intercept are adjusted to give the best alignment between the two histograms. The best matching slope and intercept values are then applied to the second file, which completes the calibration process.  **

**The automated peak finding routine performs several operations on calibrated histograms: sharp spikes in the histogram are eliminated by smoothing; using the first derivative of the histogram, the top of each peak is found and reported as the fragment size.  The smoothing and derivative steps are performed by the Savitsky-Golay kernal convolution mechanism.  Overall the routine does very well at quickly and objectively finding the crests of well defined peaks, but it tends to assign some false peaks where the histogram is noisy.   **

**RFLP + GELase + Staining + Instrument Reproducibility Study**

**Fragment size comparison among Virtual Digest, PFGE, and HSFCM of *****Staphylococcus aureus***** Mu50 (sequenced strain)**

**2  min**
**10 min**
**120 min**
**60 min**
**20 min**
**40 min**
**120 min**
**60 min**
**2x10 min**
**<8 hrs **

**Cell Culture**

**Wash**

**Form Plug**

**Lysis**

**Proteinase K**

**PMSF **
**Inactivation**

**Wash**

**GELase**

**Restriction Enzyme Digestion**

**Flow Cytometry**

***Bioscience******	***

**I-086**