Bacteria genome fingerprinting by flow cytometry.
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 subsequently 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 <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%.


CONCLUSIONS
With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.
Huang
Z
Z
Chemical Science and Technology Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Jett
J H
JH
Keller
R A
RA
D015373:Bacterial Typing Techniques; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry / Q000379:methods*; D016680:Genome, Bacterial; D011073:Polyamines / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics
D004269:DNA, Bacterial; D011073:Polyamines; D013096:Spermine; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D013095:Spermidine