Amplification of some PCR targets can be problematic(1)
. Without hot-start PCR, nonspecific priming and primer-dimer formation can occur when the reaction is below the optimal primer annealing temperature (e.g., when assembling reactions and during initial ramping of the thermal cycler). These side reactions may lead to secondary amplification products, lower yields and reduced sensitivity.
GoTaq® Hot Start Green and Colorless Master Mixes contain GoTaq® Hot Start Polymerase(4)
, which reduces the nonspecific priming and primer-dimer formation by minimizing polymerase activity until the reaction is heated to a temperature that supports specific priming. It uses antibody inhibition of Taq DNA polymerase as the hot-start method. Activating the GoTaq® Hot Start Polymerase and denaturing the anti-Taq DNA polymerase antibodies require a 2-minute initial denaturation at 94–95ºC. GoTaq® Hot Start Polymerase has characteristics similar to standard Taq DNA polymerase with an extension rate of 1 minute per 1kb of target DNA, a 5´ to 3´ exonuclease activity and lacking a 3´ to 5´ exonuclease activity.
The 2X Master Mixes contain GoTaq® Hot Start Polymerase supplied in 2X GoTaq® Reaction Buffer (either Green or Colorless), 400µM dNTPs, and 4mM MgCl2. The GoTaq® Hot Start Green Master Mix (Cat.# M5122) contains a blue and yellow dye plus a substance that increases density so that reactions can be directly loaded onto gels. The GoTaq® Hot Start Colorless Master Mix (Cat.# M5132) should be used when downstream applications require fluorescence or absorbance readings without prior purification(4)
Hot-Start PCR Performance
Amplification of some targets can be improved by using hot-start PCR. To illustrate this, we compared the GoTaq® Hot Start Green and Colorless Master Mixes with a master mix containing standard Taq DNA polymerase in amplifications of five targets that typically require hot-start PCR. For this comparison, all reactions were assembled at room temperature and placed in a room-temperature thermal cycler before starting the cycling protocol. The standard Taq DNA polymerase amplifications showed secondary products, primer-dimers, low or no yield, or a combination of these. Both GoTaq® Hot Start Master Mixes amplified the target of interest with good yield and minimal secondary products or primer-dimer formation (Figure 1).
Set Up Reactions at Room Temperature
Room-temperature reaction setup is more convenient than setup on ice for manual hot-start methods. GoTaq® Hot Start Master Mixes allow for room-temperature setup and can even stand at room temperature for 24 hours before starting the cycling procedure. The GoTaq® Hot Start Polymerase in the master mixes remains inactive during this time. To demonstrate this property, we amplified a 1.5kb fragment of the Corynephage omega gene from a plasmid template using the two GoTaq® Hot Start Master Mixes and a master mix with standard Taq DNA polymerase. Two sets of reactions were assembled. One set of reactions was amplified immediately, and the second set sat on the lab bench at room temperature for 24 hours before being amplified. As seen in Figure 2, amplifications using the GoTaq® Hot Start Green or Colorless Master Mixes gave good amplification of the 1.5kb product with no secondary products whether the reactions were cycled immediately or left at room temperature for 24 hours. Amplifications using the master mix with standard Taq DNA polymerase gave secondary products and less yield of the 1.5kb product regardless of starting conditions.
We have amplified fragments from various targets including human genomic DNA, lambda and plasmid DNA (Figure 1) using the GoTaq® Hot Start Master Mixes. We have successfully amplified targets as small as 115bp and as large as 3.1kb as well as a multiplex amplification with 6 products (Table 1, data not shown).
Table 1. Compatibility of GoTaq® Hot Start Green and Colorless Master Mixes with Various Applications.
Amplifications with the GoTaq® Hot Start Green and Colorless Master Mixes give similar yield and sensitivity. We amplified a 1.3kb β-globin fragment from human genomic DNA using both GoTaq® Hot Start Master Mixes. Different amounts of template DNA produced approximately equal yield with similar sensitivity (Figure 3).
Characteristics and Applications
GoTaq® Hot Start Polymerase adds a single deoxyadenosine to the 3´ ends of DNA in a template-independent fashion. This allows for simple T-vector cloning of the PCR fragment following purification (Table 1). In addition, the GoTaq® Hot Start Master Mixes can be used for the PCR step in uncoupled RT-PCR after synthesizing cDNA using either the ImProm-II™ Reverse Transcription System (Cat.# A3800) or the Reverse Transcription System (Cat.# A3500; data not shown and Table 1).
The Advantages of GoTaq® Hot Start Master Mixes
The use of the antibody inactivation of Taq DNA polymerase in the GoTaq® Hot Start Master Mixes has advantages over other hot-start methods. Chemical modification of Taq DNA polymerase requires a long initial denaturation (5–15 minutes) to restore polymerase activity unlike the shorter, 2-minute initial denaturation time for the GoTaq® Hot Start Polymerase. Manual hot-start methods can easily lead to contamination, are inconvenient to set up and may not work as well as other hot-start methods.
To compare competitor Taq DNA polymerase hot-start products, we amplified a 115bp HIV-1 gag fragment (4) in a human genomic DNA background using the manufacturer’s instructions. Amplification of this target requires hot-start PCR. GoTaq® Hot Start Green Master Mix was compared to the direct-to-gel loading hot-start master mix from Competitor I. This competitor's product also uses the antibody method of Taq DNA polymerase inhibition. The GoTaq® Hot Start Green Master Mix performed well with good yield, specificity and sensitivity, while the competitor’s product gave lower yield (Figure 4, Panel A). GoTaq® Hot Start Colorless Master Mix was compared with two competitor products. The product from Competitor I used the antibody inhibition hot-start method while the product from Competitor A used the chemical modification hot-start method. The GoTaq® Hot Start Colorless Master Mix performed well giving good yield, specificity, and sensitivity while the competitor’s products gave lower yields (Figure 4, Panel B).
GoTaq® Hot Start Master Mixes provide a convenient method to amplify targets and improve specificity, sensitivity and yield for some amplifications. These two products provide the convenience of master mix format, room-temperature setup and a short initial denaturation to restore polymerase activity. Assembled reactions can sit at room temperature for up to 24 hours before starting the thermal cycling procedure, which could be important for scientists using high-throughput or robotic platforms. The cycling and reaction components are the same as the familiar GoTaq® Master Mixes. To assemble reactions, you only need to add template, primer and water. As for all the GoTaq® Amplification family products, you can choose the convenience of loading directly onto gels using the GoTaq® Hot Start Green Master Mix, or choose the GoTaq® Hot Start Colorless Master Mix if the dyes may interfere with your downstream applications.