An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA.
Existing methods for extraction and processing of large fragments of bacterial genomic DNA are manual, time-consuming, and prone to variability in DNA quality and recovery. To solve these problems, we have designed and built an automated fluidic system with a mini-reactor. Balancing flows through and tangential to the ultrafiltration membrane in the reactor, cells and then released DNA can be immobilized and subjected to a series of consecutive processing steps. The steps may include enzymatic reactions, tag hybridization, buffer exchange, and selective removal of cell debris and by-products of the reactions. The system can produce long DNA fragments (up to 0.5 Mb) of bacterial genome restriction digest and perform DNA tagging with fluorescent sequence-specific probes. The DNA obtained is of high purity and floating free in solution, and it can be directly analyzed by pulsed-field gel electrophoresis (PFGE) or used in applications requiring submegabase DNA fragments. PFGE-ready samples of DNA restriction digests can be produced in as little as 2.1 h and require less than 10(8) cells. All fluidic operations are automated except for the injection of the sample and reagents.
Mollova
Emilia T
ET
U.S. Genomics, Woburn, MA 01801, USA.
Patil
Vishal A
VA
Protozanova
Ekaterina
E
Zhang
Meng
M
Gilmanshin
Rudolf
R
D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field / Q000295:instrumentation* / Q000379:methods; D005285:Fermentation; D005456:Fluorescent Dyes / Q000737:chemistry; D008904:Miniaturization; D009693:Nucleic Acid Hybridization / Q000379:methods*
D004269:DNA, Bacterial; D005456:Fluorescent Dyes