Isolation of nucleic acids is the first step in most molecular biology studies and recombinant DNA techniques. A large number of procedures, based on a variety methods and principles, exist for the extraction and purification of nucleic acid. They all share the common requirements that the biological material needs to be lysed, cellular nucleases must be inactivated, and finally, the desired nucleic acid has to be purified from the cellular debris. Roche chose a chemical/enzymatic approach for cell lysis and nuclease inactivation, and adsorption chromatography for nucleic acid purification.
The kit contains 5 different buffers, Proteinase K, High Pure tubes with pre-packed silica filters and collection tubes. The system can be used to extract nucleic acids from a wide range of sample material, including whole blood, solid tissue and mammalian cells. Protocols differ only in the first few steps regarding tissue and cell lysis; they then share a common purification procedure. Briefly, the starting material is lysed by incubation with Lysis Buffer and Proteinase K to break open cell membranes and expose DNA and RNA. Binding Buffer is added to inactivate nucleases and the solution is transferred into filter tubes and briefly centrifuged. After three washes with different buffers, the nucleic acid bound to the glass fiber filter is pure and can be eluted with the Elution Buffer into a sterile 1.5 microcentrifuge tube. Eluted DNA can be stored at 4°C, frozen at -20°C or used right away for further experiments. Elution Buffer does not interfere with further reactions. An additional incubation with RNase is optional, but in my experience, it is not required for standard DNA amplification.
The standard protocol is fast, particularly given its relative complexity and the delicacy of the procedure. Using so many buffers can be a bit confusing and prone to errors, but it is a common feature in this kind of kit where different materials have to be removed thoroughly from nucleic acid. The possibility of processing different kinds of samples with the same kit is a feature I really appreciate. Our previous system allowed extraction from pelleted cells only and the procedure was rather uncomfortable. That is why we appreciate the flexibility and quickness of the High Pure PCR Template Preparation Kit so much. Although most DNA extraction systems available today work with a wide range of samples, sometimes wider than that offered by this Roche kit, the High Pure PCR Template Preparation Kit will cover most of the needs in labs where biological samples other than blood and blood cells are rarely processed, as in our own.
I feel very comfortable with the High Pure PCR Template Preparation Kit. It is fast and accurate, has good yields and the purification quality is very high. It suits nearly all of my needs, as I extract mainly from blood or pelleted cells. In fact, its only drawback could be that it works with a relatively limited range of sample types compared to competing systems. But I think this would be an issue only for a limited number of researchers who routinely work with many different kinds of samples. Overall, Roche offers a great product, whose strengths are its ease of use, speed and the ability to process a variety of materials.
Dr. Andrea Galli
University of Milan
Dept of Clinical Sciences