Nucleic acid purification is often an annoying part of molecular biology research. It is a sort of forced stop that we must take in order to go on with our experiments, something that slows us down but is, nonetheless, necessary. It is also something that must be done properly. Filter-based purification systems offer some of the quickest and most reliable methods for nucleic acid clean-up. The High Pure PCR Product Purification Kit from Roche is one such system, using glass fibers pre-packed into a filter tube which selectively bind DNA or RNA. Nucleic acids have to measure from 0.1 to 50 kilobases in order to be retained by the filter. If they are shorter, they will be washed off with primers, salts and nucleotides. On the other hand, fragments longer than 50 kb cannot pass through the filter and will not be eluted and recovered at the end of procedure.
The High Pure PCR Product Purification Kit contains three different buffers, High Pure filter tubes and collection tubes. The binding buffer is mixed with PCR solution after amplification and transferred into a High Pure tube to allow DNA (or RNA) binding to the filter. After a short centrifugation step, bound DNA is washed twice with wash buffer. The filter tube, now containing purified DNA, is then transferred into a clean 1.5 ml microcentrifuge tube. Elution buffer is added to the upper reservoir and a quick centrifugation transfers the unbound DNA directly into the microcentrifuge tube. Eluted DNA can be stored at 4°C, frozen at -20°C or used right away for further experiments. The elution buffer does not interfere with further reactions.
The procedure is quite fast and straightforward, even when working with many samples. Nevertheless, I do not feel very comfortable using many different buffers. I think it is easier to make mistakes and ruin hours of work. Moreover, I felt the tubes contained in the kit were a bit uncomfortable to handle. Specifically, my colleagues and I found them hard to open and close. I agree it is a strange comment, but that is how we felt about them. Instead of being able to open a tube with just the thumb, it is often necessary to hold it with two hands and take much care to open it without spilling the contents. I do not know if Roche has changed the tube design in the meanwhile, but this problem was very annoying to us when working with the High Pure Kit.
At any rate, the system provides a very good yield and the purification efficiency is excellent, even when starting with low quantities of DNA. In addition, the High Pure PCR Product Purification Kit is able to purify DNA from agarose without any additional requirements. I have never used this feature, but I think it is quite useful if gel extraction is occasionally required. In that case, a new kit is not necessary and the everyday, well established, purification procedure can be used with almost no modifications.
The High Pure PCR Product Purification Kit is definitely a good product, providing a very fast purification system with a good yield. It has some drawbacks concerning the ease of use, but it could be a matter of personal taste and they do not pertain to the system reliability, which remains very high.
Dr. Andrea Galli
University of Milan
Dept of Clinical Sciences