Molecular Genetics

Advanced Biotechnologies has extensive experience in the production of a variety of human and animal viruses. Selected viruses have been purified and concentrated and are available from frozen inventory for use in genomic isolation, viral protein analysis, or other molecular virology techniques or services.

The molecular genetics services available at ABI start from basic techniques such as plasmid purification, insert isolation, and proceed to large-scale contract fermentations with downstream processing or custom isolation of viral nucleic acids. These services utilize quality-tested reagents and are the same techniques used for in-house production of new products currently being developed at ABI.

Please contact ABI for service pricing and scheduling.

DNA Probe Production and HPLC Purification of DNA Fragments

Fermentation

DNA probe production at ABI is achieved from recombinant plasmids produced in E. coli. Fermentation capacity at ABI is 125 liters in individual shaker flasks (maximum batch size). Broth media is produced in the ABI clean room facility and performance/sterility tested prior to use. The products of fermentation are pooled and harvested by continuous-flow centrifugation on a CEPA Z-41G centrifuge or Electronucleonic model RK centrifuge. If the investigator has conditions optimized in shake flasks, these conditions can be rapidly duplicated in our shaker system for rapid scale-up.

Downstream Processing

The bacterial paste is typically processed using a modified alkaline lysis and cleared lysate procedure. The crude lysate is further purified by CsCl banding to eliminate chromosomal and RNA contaminants. Typical batches have produced 0.5 gram of purified plasmid per 125 liters of culture (yield is highly dependent on construct and host bacteria).

Large-Scale Restriction Digestion of Plasmid

Use of restriction enzyme is typically optimized for completion of digest and lack of detectable nuclease activity. Conditions are optimized on a case-by-case basis.

HPLC Purification of DNA Fragments

The current scale of HPLC is 60 mg of starting DNA per run (can be scaled further). The material is chromatographed and characterized by agarose gel electrophoresis. The current separations isolate a 7kb DNA insert fragment from fragments of 1.2kb and smaller. The ability to resolve fragments is determined on a case-by-case basis. No losses are detectable for this process scale work. Current batch size is 50 mg of final purified insert product. Purity level is specified by the customer, and the current specification is less than 1% contamination by the next smaller size fragment, which has been readily achievable.