Hot start PCR

Kellogg, D. E., Rybalkin, Y., and Chen, S. (1994) Taq-start antibody hot start PCR facilitated by a neutralizing monoclonal antibodies directed against Taq DNA polymeia e Biotechniques 16, 1134-1137. 

Use hot-start tubes and assemble the bottom and top part of the reaction for second-strand synthesis and amplification of the DNA template by error-prone PCR. Hot-start PCR is the PCR technique of assembling the reaction mixture at a temperature that is greater than the annealing temperature. This procedure increases precision, yield, and specificity. The pre-adhered wax bead assures synchronous reaction start-up and eliminates the need for using mineral oil. 

In one report, anti-Taq DNA polymerase antibody was employed to avoid loss of PCR efficiency. The antibody inhibited the Taq polymerase before PCR reagents attained a high temperature, and this procedure is thus called hot-start PCR. In this procedure, the loss of Taq polymerase due to non-specific binding was reduced. This on-chip hot-start PCR resulted in a more consistent and higher yield than that obtained in the PCR chip without hot-start, and even that in the conventional PCR reaction tube (with hot-start) [917],... 

Hot start PCRs can also greatly enhance PCR specificity.,0>n In this procedure, one of the critical components for amplification (commonly Taq polymerase) is added to the tube only after the reaction temperature reaches above the Tm (melting point) of the primers. This allows for primers that annealed nonspecifically at the lower temperatures to be melted off prior to addition of Taq polymerase. As a consequence, unique amplification products can often be obtained from low copy number targets that are present in a vast background of genomic DNA. 

Today, hot-start PCR is most common technique used to reduce nonspecific amplification due to the assembly of amplification reactions at room temperature or on ice. At room temperature, PCR primers can anneal to template sequences that are not totally complementary. Since thermostable DNA polymerases have activity at these low temperatures, the polymerase can extend misannealed primers. This newly synthesized DNA is 100% complementary to the DNA template, allowing primer extension and the polymerization of undesired amplification products. However, if the reaction is heated to temperatures above 60°C before polymerization begins, the stringency of primer annealing is increased, and subsequent synthesis of undesired PCR products is avoided or reduced. 

Hot-start PCR can also reduce the amount of primer-dimer formation by increasing the stringency of primer annealing. At lower temperatures, the primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer-dimer, which can often be observed as a diffuse band of approximately 50 to 100 bp on an ethidium bromide-stained gel. The formation of nonspecific amplification products and primer-dimer can compete for reagent availability with the amplification of the product desired. Therefore, hot-start PCR can improve the yield of the specific PCR products. 

A typical reaction mixture (100 jul) contains 20 mM Tris—Cl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.2 mM each of four dNTPs, 2 ng DNA, and 0.1 /xM of each primer. The mixture may optionally include 5% (v/v) Me2SO, 0.01% (v/v) gelatin, and/or 0.05% (v/v) NP-40/Tween 20. The polymerization reaction is initiated by adding 2.5 U Taq Pol. [Note In hot-start PCR (see further below), the polymerase is made to come into contact with the rest of the mixture at or above the primer annealing temperature (5). To prevent evaporation during PCR, the reaction mixture is overlaid with two or three drops of mineral oil (e.g., Nujol) or one pellet of paraffin wax (e.g., AmpliWax of Perkin-Elmer)]. 

The samples are then overlaid with mineral oil (20 pi) to prevent evaporation of the contents and the PCR can proceed. If, however, the PCR machine being used has a hot-start lid, addition of mineral oil is not necessary. 

Equipment PCR machine, scintillation counter, tabletop centrifuge, temperature-controlled water baths, equipment for horizontal and vertical electrophoresis, UV-illuminator, phosphor imager, automatic DNA sequencer, vacuum dot-blot manifold (Schleicher and Schuell). PCR 0.5 ml hot-start mbes, aerosol resistant pipette rips, autoclaved Eppendorf tubes (all from Fischer Scientific, Brightwaters, NY) and glassware, diethyl pyrocarbonate (DEPC, Sigma)-treated solutions. 

Hot Start. To minimize nonspecific PCR, hot start can be incorporated into the amplification protocol." Reaction mixes containing DNA template, primer pair and dNTP in PCR buffer are heated to 80°, and an aliquot of diluted Tag polymerase is then added to the mixture to initiate the amplification reaction. 

Samples that have very low target mRNA concentrations often display increased background, particularly if amplification above 35 cycles is required. The initial formation of these products can occur at low initial (ambient) temperatures, and are reduced or eliminated by the hot start approach, in which wax beads are added, melted to form a solid layer above the cDNA, and then the PCR master mix added on top. The two solutions are mixed and the PCR reaction begins after the thermocycler heats to 94°C. Background is sometimes due to the formation of primer dimers, a double-stranded PCR product consisting of the two primers and their complementary sequences. Sometimes these primer dimers contain extra sequences between the primers (14). When primer dimers form, they can be a major problem as they are very efficiently amplified and compete with amplification of the target cDNA. This is not a major problem with the primer sets described in Table 1, but can be a problem with other primer sets. With persistent problems, the hot start approach could be modified to add the cDNA, the primers, and the Taq polymerase separately. 

The usual criteria for maintaining probe specificity during PCR amplification apply, such as low concentration of target nucleic acid ( 0.5 ng of a plasmid), limitation of the number of cycles (an excess of cycles rapidly increases the amount of nonspecific probes), hot-start of PCR to avoid nonspecific annealing and extension of primers to nucleic acid at low temperature (i.e., nucleic acid or primer or enzyme are added at high temperature). 

Use same cycle parameters as first-round PCR, but use a hot start. After the final cycle, polish the second-round PCR products by adding 50 ng of each primer, 0.5 pL of dNTPs, and 0.5 pL of Amplitaq. Perform one cycle using the following conditions ... 

In a thin-wall microcentrifiige tube (r f Note 5), mix the following 5 pL of 5x Phire Hot Start II Polymerase buffer see Note 6), 2.5 pL of lOx dNTPs for PCR, 1 pL of gene specific forward primer (10 pmol/pL see Note 7), 1 pL of cDNA from Subheading 3.1, step 4, and water to bring the volume to 24.5 pL. 

Our laboratories routinely use the Phire II Taq DNA Polymerase for PCR amplifications. However, we have used other enzymes, hot-start and not, on occasion. Thus, the choice of enzyme (and associated buffers) is largely a matter of the preferences of users. 

---