Several approaches are available that can improve upon the specificity and sensitivity of various PCR applications. Some of these approaches utilize specialized reagents, while others employ modified PCR cycling parameters or variations of oligonucleotide primer design.
5.1. IMPROVED TAQ POLYMERASE PREPARATIONS Several specialized PCR reagents have emerged over the last decade that function to improve PCR specificity. These include special preparations of Taq polymerase. Taq polymerase has substantial polymerase activity at 37°C, even though its optimal activity is expressed at much higher temperatures (approx 72°C). This low-temperature polymerase activity is the basis for some mispriming events that occur during the initial phase of PCR. Extension can occur from oligonucleotide primers that anneal nonspecifically to template DNA prior to the first denat-uration step at 93-95°C. Platinum Taq Polymerase (from Invitrogen Life Technologies, http://www.invitrogen.com) was introduced to combat this problem by including a thermolabile inhibitor of the Taq polymerase in the form of monoclonal antibodies. During the initial denaturation phase of PCR, the elevated temperature destroys the monoclonal antibodies, releasing the Taq polymerase to function in PCR. The antibody-mediated inhibition of Taq allows for room-temperature reaction assembly. TaqBead Hot Start Polymerase is a related product from Promega (http://www.promega.com). In this preparation, the Taq polymerase is physically sequestered within wax beads. The Taq polymerase becomes mixed with the PCR mixture during the initial denaturation step of PCR when the wax bead melts (at approx 60°C). In both cases, the Taq polymerase is functionally inactive until a critical temperature is reached, eliminating or reducing the possibility of primer extension from mispriming events.
5.2. HOT-START PCR Hot-start PCR was developed to reduce background from nonspecific amplification by preventing polymerization of new DNA during the initial phase of the reaction, when nonspecific binding could occur between primers and other DNAs in the mixture (28,29). Hot start could be achieved by limiting the initial concentration of one or more PCR mixture components, including MgCl2 or dNTP. However, the more commonly applied version of hot-start PCR separates the components of the PCR mixture with a barrier that is removed when the PCR is initiated. Wax beads form the basis for the typical physical barrier employed in this application. Critical components of the reaction, including Taq polymerase, are separated from the remainder of the reaction by the wax barrier, which melts as the mixture is heated. The key concept in hot-start PCR is to achieve a temperature greater than the annealing temperature of the oligonucleotide primers prior to the complete mixing of the reaction components initiating polymerization.
5.3. TOUCH-DOWN PCR Touch-down PCR was developed to enhance amplification of desired target sequences while reducing amplification from mispriming events or from other PCR artifacts (30,31). In this approach, the initial PCR cycle begins with an annealing temperature that is greater than the calculated Tm of the oligonucleotide primers utilized. The elevated annealing temperature employed during early cycles will encourage high specificity of primer annealing, although at lower efficiency. In subsequent cycles, the annealing temperature is progressively and incrementally lowered, improving the efficiency of oligonucleotide primer annealing. By manipulating the annealing temperature over the course of PCR, the desired amplicon will preferentially accumulate with good yield while amplification of undesirable products is minimized.
5.4. IMPROVING PCR SENSITIVITY Polymerase chain reaction is very sensitive and detectable levels of amplified products can usually be produced in a standard reaction consisting of 25-30 cycles. However, given the complexities of the factors that determine yield of PCR products (including the starting concentration of target sequence within the template and the efficiency of the reaction), there will be times when a standard reaction will not yield sufficient product to be visualized or otherwise detected. There are several strategies to overcome this problem. The simplest solution is to perform a second round of PCR using a small quantity of the first-round reaction mixture as the template source. In most cases, 25-30 additional cycles of PCR using this target-enriched template will result in detectable product. A second solution to this problem is nested primer PCR. In this application, the first-round PCR is accomplished using one set of primers, and the second-round PCR is accomplished using oligonucleotide primers that are target-sequence-specific, but sited internal to (or nested within) the first oligonucleotide primer pair. Thus, the first-round of PCR provides a target-enriched template for the second-round of amplification using the nested primers. The advantage of nested PCR over a simple second-round PCR involves the specificity of the amplification reactions. By using distinct PCR primers that are directed to the same target sequence, nested PCR maintains a high level of specificity in the second round of amplification. Nonspecific products produced during the first round of PCR are not likely to contain sequences complementary to the nested primers. Thus, spurious first-round amplicons are eliminated during the second round of PCR. In contrast, second-round amplification using the same PCR primers is less stringent and will result in amplification of both the desired template as well as any nonspecific products from the first reaction. In addition, extremely rare target sequences can be detected using nested PCR. The improved sensitivity of nested PCR is related to the fact that the first round of PCR effectively enriches the template sample prior to the second round of PCR. Because of the sensitive nature of nested PCR, special care must be taken in order to avoid contamination.
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