HotStart-IT™ Binding Protein is the active component in a novel hot start technology 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, we have 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.

Properties

• HotStart-IT Binding Protein performs well in many standard PCR reaction buffers.

Purity

• Free from detectable non-specific nucleases.

Storage Buffer

• 20 mM Tris-HCl (pH 8.5), 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol.

Mass Definition

• In general, one microgram of HotStart-IT Binding Protein sequesters about 5 pmol of primers.

Concentration

• 10 ± 0.5 mg/mL

Source

• Recombinant protein expressed in E. coli.

Application

• Hot-Start PCR Amplification

Advantages of HotStart-IT

• Room temperature reaction set-up
• High specificity and sensitivity
• Minimizes amplification of non-specific products and primer-dimers (Fig. 2)
• Ideal for complex templates and multiplex reactions
• Unlike chemically-modified Taq, no extensive heating step is necessary which may damage precious samples
• Technology is portable to a polymerase of choice.

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 (10 mM Tris-HCl [pH 8.6], 50 mM KCl, 1.5 mM MgCl₂), 0.2 mM 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. (See Fig. 2 for representative data.)

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.