HotStart-IT Binding Protein
HotStart-IT® Binding Protein is a recombinant protein expressed in E. coli.
Hot-Start PCR Amplification
HotStart-IT Binding Protein is the active component in a novel hot start technology developed at USB called primer sequestration. In general, hot start PCR methods reduce or eliminate non-specific primer-extension products formed at lower temperatures during assembly of PCR reactions. At these less stringent annealing temperatures, primers may bind non-specifically, which often leads to unwanted amplification products and primer-dimers. In order to resolve this problem, USB has produced a high-quality DNA binding protein that is especially useful at sequestering primers at lower temperatures making them unavailable for use by a polymerase (Figure 1). This primer-sequestration technique effectively blocks DNA synthesis from mis-priming events at lower temperatures. Following the initial denaturation step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. Unlike other hot start methods, such as antibodies or chemical modifications, the binding protein is compatible with a variety of thermostable polymerases. HotStart-IT Binding Protein has been designed for PCR applications that demand extremely high specificity and sensitivity and is thoroughly tested for purity and performance.
HotStart-IT Binding Protein performs well in many standard PCR reaction buffers.
Free from detectable non-specific nucleases.
20mM Tris-HCl (pH 8.5), 200mM KCl, 0.1mM EDTA, 1mM DTT, and 50% glycerol.
In general, one microgram of HotStart-IT Binding Protein sequesters about 5 pmol of primers.
Quality Control Polymerase Blocking Assay:
This assay compares the amount of Taq DNA Polymerase activity with 2 μg HotStart-IT Binding Protein against its activity with no binding protein. The reaction mixtures contain 0.625 units of polymerase, 1X PCR Reaction Buffer (10mM Tris-HCl (pH 8.6), 50mM KCl, 1.5mM MgCl2), 0.2mM each dNTP, and 2 pmol of overlapping, extendable oligonucleotides in a 25 μl reaction volume. Following incubation at 25°C for 4 hours, HotStart-IT Binding Protein blocks at least 90% of the Taq DNA Polymerase activity.
|Figure 2. Increased specificity due to HotStart-IT Binding Protein. Single-copy numb and p53 genes were amplified from the indicated amounts of human genomic DNA using standard Taq DNA Polymerase without (-) and with (+) 2 μg of HotStart-IT Binding Protein per reaction. The primers in this assay were designed with 3 bases of overlap at their 3'-ends to favor primer-dimer formation during reaction set-up at room temperature. Results demonstrate a shift from mainly primer-dimers to the desired products when the binding protein was included.|
Tested User Friendly™ Functional Test:
PCR with Taq DNA Polymerase and HotStart-IT Binding Protein shifts production of primer-dimers to a specific target of 306 bp from 1 ng of human genomic DNA relative to Taq DNA Polymerase alone.
Shipped on dry ice. Store at -20°C