As the “gold-standard” for quantitative DNA analysis has become quantitative Real-Time PCR, the need for reliable, cost-effective methods of analysis have become paramount. The ABgene ABSOLUTE™ QPCR SYBR® Green Mix is a robust, relatively inexpensive product, which allows quantitative template analysis by Real-Time PCR.
The premise of the ABgene SYBR® Green Mix is the same as other SYBR® Green mixes; namely, the reaction mix contains a dye (SYBR® Green) which is non-fluorescent when unbound to DNA, but when incorporated into double-stranded DNA strands, as during PCR amplification, the fluorescence it emits is readily detectable by Real-Time PCR thermal cycler systems. In the linear phase of the PCR reaction (where the amount of DNA present doubles for every one cycle of the reaction), the relative quantity of DNA in each sample can be measured by comparing the level at which the SYBR® Green fluorescence curves cross an arbitrary threshold. This system is far superior to standard PCR methods, using “bands on a gel” and densitometry.
The ABgene SYBR® Green Mix varies from other such mixes in that it contains a patented, chemically-modified DNA polymerase called Thermo-Start®. This polymerase is inactive until being “switched on” by incubation at high temperature. ABgene recommends a 15 minute preliminary incubation step at 95oC in the first cycle of the PCR reaction; in practice, this only increases the PCR run-time by 10-12 minutes, as the first step in most standard reactions is a 3-5 minute incubation step at 95oC to denature DNA. The ABgene SYBR® Green Mix also comes with ROX dye: a fluorescent “standard” to compensate for pipetting errors and allow normalization between samples; it can be added separately. In practice, I have never used this dye as a reference; using a well-chosen housekeeping gene provides a biologically-relevant method of normalization between samples, although in practice the use of ROX may be a good idea for some applications. Additionally, ABgene claims the reaction buffer in the SYBR® Green Mix provides higher sensitivity and specificity than other buffers, although no details are provided in the specification sheet. A vial of MgCl2 is supplied, allowing optimization of reactions for specific primer-template combinations, although personally, I have never needed to use it.
In summary, the ABgene QPCR SYBR® Green Mix is cheaper (by about 20%) than other comparable SYBR® Green mixes I have used, and the results are equally specific, sensitive and reproducible. Additionally, as the mix is stored at -20oC, we have found that once the PCR reaction is set up (including primers, template and SYBR® Green Mix), it can be conveniently frozen at -20oC for at least one week, with no discernible differences in subsequent quality of the PCR reaction.
Graduate Student
Research Department
Peter MacCallum Cancer Centre