I have used the 5 PRIME HotMasterMix for the PCR amplification of open reading frames (ORF) from different pathogenic microorganisms followed by cloning and in vitro expression in a T7 expression vector. Following expression, the expressed proteins are printed onto a microarray chip to screen for antigens. This type of high throughput analysis requires a great deal of accuracy. Initially, we used standard Taq DNA polymerase and made the master mix ourselves, on ice, to maintain enzyme activity and decrease non-specific amplification. In addition, prior to making the master mix, each component of the PCR reaction (i.e. dNTPs, gelatin, PCR buffer, Taq polymerase and Mg++) was thawed individually. The large number of handling steps required to run a PCR reaction made it very likely to make mistakes.
After receiving a free sample of 5 PRIME HotMasterMix, I did a complete 96 well plate reaction and found that I had greater than 90% success. Agarose gel electrophoresis confirmed that the amplification products were the correct size (based on the DNA ladder) and there were no non-specific bands. Compared to traditional PCR using Taq, the intensity of the bands was less for the 5 PRIME HotMasterMix. I then cloned the genes into a vector and subsequently sequenced them, only to find greater than 90 percent success. Better quality can be obtained by sacrificing some PCR product yield, but if more product is required then the number of cycles can be increased.
We had been working with many different pathogenic microorganisms, thus some had unique sequence compositions in the genome, making amplification more difficult using the traditional Taq enzyme. With HotMaster mix, the only thing you need to do is to add 10% DMSO or 1 M Betaine (for GC rich templates) and decrease the extension temperature (from 65„aC to 50„aC-55„aC) for AT rich templates to get very good amplification. Furthermore, the magnesium concentration does not need to be adjusted as it is pre-optimized and self-adjusting (via weak chelation of the Mg ion). With HotMaster mix, the only requirement for PCR is the combination of DNA template, primers, HotMasterMix, and molecular biology grade water, a quick spin and placement in the PCR machine. During the liquid handling period, you can work at room temperature, making it an advisable choice for researchers who work with high-throughput PCR and DNA cloning. This system enables more time for analyzing data, rather than liquid handling protocols.
Yuan Zhong
Graduate Student
Department of Molecular Biology and Biochemistry
University of California, Irvine
At the time of this review the HotMasterMix was commercialized by Eppendorf. Today it is available from 5 PRIME. This review was edited to reflect this fact.