An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA

作者：Mollova Emilia T; Patil Vishal A; Protozanova Ekaterina; Zhang Meng; Gilmanshin Rudolf^*

来源：Analytical Biochemistry, 2009, 391(2): 135-143.

DOI：10.1016/j.ab.2009.05.008

• Sample preparation
• Automated mini-reactor
• Submegabase DNA fragments
• Bacterial genomic DNA
• Probe hybridization
• Fluorescent tagging

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 108 cells. All fluidic operations are automated except for the injection of the sample and reagents.

• 出版日期2009-8-15