Real-time quantitative PCR is a powerful tool to detect and quantify nucleic acids. By incorporating fluorescently labeled probes or nucleic acids or fluorescent double-stranded DNA (dsDNA)-binding dyes into the PCR, product formation can be monitored following each PCR cycle. There are many advantages to using real-time PCR over endpoint PCR. Use of fluorescent dyes to monitor amplification eliminates the need for gel electrophoresis, saving the user time and reducing the potential for contamination. Since product formation can be detected in the exponential phase of PCR where the reaction is most efficient, quantification of starting material is more accurate and sensitive (1). The sensitivity offered by fluorescent dyes also allows detection over a much broader dynamic range than endpoint PCR.
Probe-based chemistries such as TaqMan® technology are advantageous in that the labeled probe, which is specific to the amplicon, produces a fluorescent signal during the enzymatic processing of the probe:amplicon hybrid, indicating the specific synthesis of amplicon. However, these assays are difficult to design and more expensive than dye-based chemistries. dsDNA-binding dyes, such as SYBR® Green I, detect all dsDNA formed during amplification; however, these assays are simple to design and cost-effective. The ability to perform melt curve analysis, which is not possible with the TaqMan® assay, provides a quality control step to verify amplification of the desired amplicon.
GoTaq® qPCR Master Mix (Cat.# A6001) introduces a new, proprietary dsDNA-binding dye, Bryt™ Green dye, that can be used at a higher concentration than SYBR® Green I because it is less inhibitory in an amplification reaction. The dye concentration in the master mix is optimized to produce significantly brighter fluorescence during qPCR than master mixes containing SYBR® Green I. Excitation and emission of the dye are similar to those of SYBR® Green I (Figure 1), so it is compatible with commonly available instrumentation platforms. GoTaq® qPCR Master Mix is a 2X master mix composed of an optimized buffer formulation complete with dNTPs and MgCl2 and features GoTaq® Hot Start Polymerase . GoTaq® Hot Start Polymerase contains full-length Taq DNA polymerase bound to a proprietary antibody that prevents polymerase activity at room temperature. Thermal activation is achieved by incubating the assembled reaction at 95°C for 2 minutes. The proprietary polymerase/buffer formulation accommodates extended cycle numbers (45–50 cycles) and is compatible with thermal cycling programs that include extended activation (95°C for 10 minutes). The master mix comes premixed with a low level of CXR reference dye, which is identical to ROX™ reference dye. The master mix is compatible with existing experimental designs and requires addition of only DNA template, target-specific primers and water.
Figure 1. Fluorescence spectra of SYBR® Green I and the proprietary Promega dye in the GoTaq® qPCR Master Mix. Fluorescence emission spectra were collected for both SYBR® Green I and Promega Bryt™ Green in the absence of dsDNA and in the presence of 50ng/µl or 200ng/µl dsDNA. Peak emission wavelength is 523nm for SYBR® Green I and 529nm for Bryt™ Green.