Bacteria genome fingerprinting by flow cytometry

✍ Scribed by Zhengping Huang; James H. Jett; Richard A. Keller

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 96 KB
• Volume: 35
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19990201)35:2<169::aid-cyto9>3.0.co;2-k

✦ Synopsis

Background: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. Methods: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subse-quently introduced into our ultrasensitive flow cytometer from a capillary. Results: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in F7 min with F2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes D20 h and requires D1 µg of DNA with a sizing accuracy of D90%. Conclusions: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.