The Advantages of Hot-Start PCR Technology

A number of non-specific priming events occur under the low stringency conditions of ambient temperature. Template hybridizes to itself, primer dimers form, and individual primers form hairpin structures or partially anneal to non-specific sites on the template. Therefore, preparing PCR at room temperature can generate secondary products in the first PCR cycle that are amplified in subsequent cycles. Even when assembled on ice, the reactions briefly pass through low stringency temperatures on the way to the first melting step. The amplification of secondary products and the non-specific exonuclease activity also unnecessarily consumes PCR reagents inhibiting the amplification of the specific desired product. Normally, using template amounts in excess of 100 to 500 copies avoids some of these difficulties. However, with lower amounts of PCR target (especially in the presence of excess non-specific and complex genomic DNA), these low rates of room temperature extension and nuclease activities affect the specificity and efficiency of the polymerase chain reaction. Skewed threshold cycle values and false amplicon melting temperatures in real-time PCR as well as false end points in conventional PCR can all occur as a result.

2. How Hot-Start PCR Helps

Hot-start technology overcomes these phenomena to generate cleaner PCR products (1). The methodology prevents non-specific extension or degradation of nucleic acid substrates at ambient temperatures by either excluding or reversibly inhibiting the polymerase enzyme. Upon assembly, pre-heating the other reaction components melts all priming events, both specific and non-specific. Addition of the polymerase, if missing, then initiates PCR. Alternatively, the heat also reverses the inhibition of the enzyme thus activating it. The first annealing step, due to its properly defined temperature, allows specific annealing reactions to occur and prevents non-specific annealing events. With a lack of non-specific hybridization of primers to template or to one another, the resulting amplified DNA bands are cleaner.

1. Manual Techniques:
   
   Manual hot-start, the simplest hot-start method, requires the researcher to withhold a critical component, usually the polymerase, until the reaction has been heated briefly at the melting temperature. Addition of the enzyme then initiates the reaction. This method proves difficult and inconvenient to perform, especially when processing many reactions at the same time, because the tubes must be kept at 100 °C in the PCR hot block, which serves as the working surface. This method also increases the risk of inadvertently contaminating the reactions.

2. Use of Physical Barriers:
   
   This relatively simple hot-start method separates the critical polymerase component from the template, primers, and other reaction components with a physical barrier that the high melting temperature removes (2-4). The most commonly and easily used barrier is wax and requires the following steps. A PCR tube containing most of the reaction components receives a molten bead of wax. Upon cooling, the wax forms a solid barrier over the aqueous phase and a receptacle for the addition of an aliquot of the polymerase. Upon reheating during the thermal cycles, the wax barrier melts, allowing the polymerase to mix with the other components in the aqueous phase.

3. Reversible Polymerase Inactivation & Specially Formulated Hot-Start Polymerases:
   

   1. Non-Covalently Bound Inhibitor:
      A polypeptide, antibody (5), or oligonucleotide aptamer (6) mixed with the polymerase binds to the active or nucleotide-binding site of the polymerase, rendering the enzyme inactive. Upon heating, the compound denatures and dissociates from the polymerase, restoring enzyme activity. The non-covalent protein-protein or oligonucleotide-protein interactions between the inhibitor and the enzyme require only relatively low activation energy, normally one to five minutes at 95 °C, to remove the inhibitor from the active site. However, the ability of the inhibitors to re-associate with the enzyme active site during thermal cycles may still disrupt or slow the reaction affecting the yield of product.
   2. Chemical Modification:
      Covalent modifications of amino acid residues in the polymerase, particularly those in the active site, also inhibit the enzyme's activities. Typical protein modification reagents each react with a specific type of amino acid. For some of these reagents, a combination of heat, water and a change in pH hydrolyzes their covalent modifications to regenerate the active amino acid, release a more inert compound, and restore enzyme activity. Unlike the dissociation of inhibitors from the enzyme, this chemical reactivation of the polymerase is irreversible because the activation process breaks molecular bonds. This process requires higher activation energy, ten to even fifteen minutes at 95 °C, than the dissociation of inhibitors further insuring the complete melting of non-specific annealing events.

3. Examples of HotStart enzymes:

The reversible inactivation of the polymerase, whether by use of an inhibitor or chemical modification, remains the most effective hot-start method. However, individual researchers lack the time or expertise to generate such enzymes routinely and consistently. Fortunately, several manufacturers offer at very reasonable prices specially formulated polymerase enzymes carefully prepared with lot-to-lot consistency. A few examples are listed below:

4. Performance of Specially Formulated Hot-Start Polymerases:

Effective hot-start polymerases should have minimal to no polymerase activity at ambient temperature and should only yield product when properly activated. Figure 1 compares a hot-start enzyme with a conventional one. Indeed, the hot-start enzyme only generates product when activated, while the conventional enzyme generates product whether pre-incubated at high temperature or not. Furthermore, the activated hot-start enzyme amplifies DNA equally as well as the treated or untreated conventional enzyme indicating that the pre-modification of the enzyme and its reversal do not affect the enzyme's proficiency. The small amount of product observed from the inactivated hot-start enzyme results from partial activation by the brief melting step in each cycle of the PCR program.

Figure 1: Activation of ReactionReady™ HotStart "Sweet" PCR master mix. The "Sweet" and HotStart "Sweet" master mixes were used to amplify a gene-specific fragment in replicate reactions that were either not activated or activated at 95 °C for 15 min. The master mixes only differ in their source of polymerase: The "Sweet" contains a standard enzyme, while the HotStart "Sweet" contains a specially formulated hot-start enzyme. Products were characterized by agarose gel electrophoresis.

Before the polymerase amplifies or degrades any nucleic acid substrate, the same heat activation process must also successfully melt the non-specific annealing and priming events. For example, primer dimers, one of the most commonly observed non-specific PCR products, occur when primer pairs complementary at their 3'-ends anneal to each other allowing primer extension from the 3'-ends to generate a small double-stranded product. The amplification of primer dimers unnecessarily consumes primers and nucleotides, frequently reducing the yield of the desired amplification product. Primer dimer formation during PCR could occur due to poor primer design or failure to use or activate a hot start enzyme. As shown in Figure 2, a conventional enzyme primarily amplifies a primer dimer at the expense of the actual gene-specific fragment. In contrast, the hot-start enzyme produces only the expected fragment of the correct size, without any primer dimer, and generates a greater amount of the product.

Figure 2: The ReactionReady™ HotStart "Sweet" PCR master mix eliminates problematic primer dimers. XpressRef™ Human Universal Reference Total RNA (GA-004, 3 µg) was converted to PCR template using the ReactionReady™ First Strand cDNA Synthesis Kit. Equal amounts of template were added to separate reactions to amplify a gene-specific fragment of human BCL10 using either SABiosciences' HotStart "Sweet" master mix or a standard non-hot start PCR enzyme. Products were characterized by agarose gel electrophoresis.

Interestingly, the length of time required for activation significantly contributes to the effectiveness of the hot-start enzyme, and the activation time of each commercially available enzyme varies. The longer the incubation time, the more likely non-specific annealing events melt and the more likely cleaner and specific products result. Figure 3 compares the ability of three different hot-start enzymes to amplify three different human genes. One enzyme relies on an antibody inhibitor and a short activation time. The other two both use chemical modification with one needing a longer activation time than the other. The results demonstrate that the hot-start enzymes with short activation times generate a population of products of various sizes for all three genes, most likely resulting from non-specific annealing of the primers to the template. However, the enzyme with the longer activation time yields predominately one band of the predicted size for the BAX and ITGA5 genes and correctly fails to yield a band in the case of the poorly expressed IL11 gene. Therefore, longer activation times allow more than enough time for non-specific annealing events to dissociate preventing the formation of secondary products.

Figure 3: The ReactionReady™ HotStart "Sweet" PCR master mix outperforms other competing hot start enzymes. XpressRef™ Human Universal Reference Total RNA (GA-004, 3 µg) was converted to PCR template using the ReactionReady™ First Strand cDNA Synthesis Kit. Gene-specific fragments of three different human genes (BAX, ITGA5, IL11) were amplified by PCR from equal amounts of template using the same primers and using either SABiosciences' HotStart "Sweet" master mix or one of two hot start enzymes from other manufacturers, according to their respective specifications. The enzyme in the HotStart "Sweet" master mix requires a longer activation time than the other two enzymes. The products were characterized by agarose gel electrophoresis.

Summary:

With the advent of hot-start technology, the polymerase chain reaction becomes an even more powerful tool for the generation and characterization of specific DNA products. Preventing the thermostable DNA polymerases from acting on non-specific annealing events avoids the production of non-specific products and enhances the yield of the desired product. The combination of PCR with the reverse transcription of RNA into cDNA template (RT-PCR) proves extremely useful for relative gene expression profiling and array data verification. However, the heterogeneity of cDNA generated from a cell's complement of total, or even messenger, RNA makes the careful design of the RT-PCR experiment even more critical to its success. The use of hot-start DNA polymerases during RT-PCR overcomes most if not all of the potential difficulties presented by the PCR phase. Thus, specially formulated enzymes, particularly those with longer activation times, are important not only for routine PCR applications but also for relative gene expression profiling by RT-PCR.

Related Products:

ReactionReady™ HotStart "Sweet" PCR master mix (P-1000B)

The ReactionReady™ HotStart "Sweet" PCR master mix from SABiosciences contains a specially formulated chemically modified version of Taq DNA polymerase for hot start PCR. This master mix has the same convenient advantages as the "Sweet" PCR master mix. To be ready for PCR, just add water, template, and primers to the lyophilized orange spheres. The orange tracking dye allows the transfer of the reactions directly from the PCR tube to the agarose gel well without the need to add gel-loading buffer first.

RT2 Real-Time™ master mix (PA-008)

The RT² Real-Time™ master mix from SABiosciences contains a specially formulated chemically modified version of Taq DNA polymerase for hot start PCR. The master mix has also been experimentally optimized for SYBR® Green compatible real-time PCR. To be ready for real-time PCR, just add water, template, primers, and SYBR® Green to the lyophilized spheres.

MultiGene-12™ RT-PCR Profiling Kits & RT² Real-Time™ Gene Expression Assay Kits:

Both of these gene expression kits combine master mixes containing hot-start PCR enzyme and carefully designed single or multiple gene-specific primer pairs for PCR.

XpressRef™ Human Universal Reference Total RNA (GA-004)

Prepared from 20 different human adult and fetal normal major organs to ensure the broadest gene coverage. Used to optimize or as a control for RT-PCR experiments.

References:

1. D'Aquila, R.T. et al. (1991) Nucl. Acids Res. 19, 3749.

2. Chou, Q. et al. (1992) Nucl. Acids Res. 20, 1717.

3. Bassam, B.J. and Caetano-Anolles, G. (1993) BioTechniques 14, 30.

4. Wainwright, L.A. and Seifert, H.S. (1993) BioTechniques 14, 34.

5. Sharkey, D.J. et al. (1994) Bio/ Technology 12, 506.

6. Dang, C. and Jayasena, S.D. (1996) J. Mol. Biol. 264, 268