A common problem with the polymerase chain reaction is the formation of non-specific products, especially prim-er-dimers. These unwanted products interfere with generating the desired amplicons. A major cause for this lack of specificity occurs during reaction set-up and before the initial denaturation step. At these lower temperatures before denaturation, primers can bind to non-target sequences with incomplete homology, such as other primers(1,). These non-specific hybrids can be extended by the polymerase, even at lower temperatures, which creates competing targets during subsequent PCR cycling (e.g. primer-dimers). Hot start PCR methods provide a solution to this lack of specificity by reducing or eliminating non-specific product formation before high-temperature cycling(2,).
Current hot start methods target the polymerase by muting its activity before the initial denaturation step, most commonly with a blocking antibody or chemical modification. Problems with these methods are two-fold: 1) the antibody is from hybridoma cells which can contaminate reactions with mammalian DNA; and 2) removal of the chemical blocking group on the polymerase typically requires initial denaturation times of greater than 10 minutes which causes heat-damage to DNA samples. An ideal solution is a hot start method that eliminates the risk of contaminating mammalian nucleic acids while also allowing shorter initial denaturation times.
To address these issues, USB Corporation has developed an alternative hot start strategy called HotStart-IT®. This novel method uses a single-stranded DNA binding protein to sequester primers at lower temperatures, making them unavailable for extension by the polymerase. Following the initial denaturation step, the binding protein is inactivated and the primers are free to participate in the amplification reaction ((Fig. 1)). This technique solves the problems that arise from other hot start methods because the binding protein is produced in E. coli and is heat-labile so that initial denaturation times are 2 minutes or less. Thus, there is no chance of mammalian genomic DNA contamination and less chance of heat-induced DNA damage. Another advantage is that because the method targets the primers and not the polymerase, it is portable to a variety of thermostable polymerases. USB has demonstrated that the “primer-sequestration” technique effectively blocks non-specific product formation before thermal-cycling and enhances end-point and real-time PCR reactions.Fig. 1.
Bottom Panel: HotStart-IT® binding protein blocks non-specific product formation at low temperatures which results in successful PCR reactions.
To validate the USB HotStart-IT® method, a 306 base pair target from the single-copy numb gene was amplified from human genomic DNA with USB Taq PCR Master Mix (without hot start) or with HotStart-IT® Taq PCR Master Mix. The standard Taq Master Mix does not contain any binding protein and, thus, has no hot start feature. Primers were designed with 3 bases of complete homology at their 3′-ends to favor primer-dimer formation. To provide the most extreme testing conditions, the reactions were set-up at room temperature and were also pre-incubated at 25°C for one hour prior to thermal-cycling to facilitate primer binding and low-temperature synthesis. As seen in (Figure 2), the results of this “failure-by-design” experiment demonstrated that when no hot start method was used, the primer-dimer created in the pre-incubation step competed with the diluted template for preferential amplification. This problem intensified as less template was used. When HotStart-IT® was used, a shift from mainly primer-dimers to the desired product occurred, which enhanced the overall specificity and yield of the reaction.Fig. 2.
Left Panel: USB hot start method results in a shift from primer-dimers to the desired target.
Right Panel: Description of reaction conditions for the experiment.
To demonstrate the functional equivalence of USB HotStart-IT® compared to current hot start methods, the previous assay was performed versus two alternative products using 1 nanogram of human genomic DNA as template. As seen in (Figure 3), all three hot start methods caused a shift from primer-dimers to the specific target compared to a reaction which used no hot start method. These data confirm that the USB hot start technique is as effective as methods that use a blocking antibody (Platinum® Taq) and chemical modification (HotStarTaq™) without the risk of mammalian DNA contamination or DNA damage.