About Polymerase chain reaction (PCR)

PCR

Basic PCR The PCR process was originally developed to amplify short segments of a longer DNA molecule (Saiki et al. 1985). A typical amplification reaction includes target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor of more than a million in a matter of hours. 

Each cycle of PCR includes steps for template denaturation, primer annealing and primer extension. 

The initial step denatures the target DNA by heating it to 94°C or higher for 15 seconds to 2 minutes. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase. 

In the next step of a cycle, the temperature is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C. 

The extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C for denaturation. 

Each step of the cycle should be optimized for each template and primer pair combination. If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. After 20–40 cycles, the amplified product may be analyzed for size, quantity, sequence, etc., or used in further experimental procedures. RT-PCR Thermostable DNA polymerases used for basic PCR require a DNA template, and as such, the technique is limited to the analysis of DNA samples. Yet numerous instances exist in which amplification of RNA would be preferred. To apply PCR to the study of RNA, the RNA sample must first be converted to cDNA to provide the necessary DNA template for the thermostable polymerase. This process is called reverse transcription (RT), hence the name RT-PCR. Reverse transcriptases (RTs) are RNA-directed DNA polymerases that were first identified as part of the retroviral life cycle (Temin and Mizutani, 1970, Baltimore, 1970). RTs catalyze the synthesis of a DNA copy (cDNA) of the target RNA molecules using a reverse transcription primer, dNTPs, and Mg2+ or Mn2+ as a cofactor. Reverse transcriptases have been adapted for use in a variety of in vitro applications including real-time and endpoint RT-PCR, labeled-cDNA probe generation and cDNA library construction. The ideal reverse transcriptase is robust (highly active under a variety of conditions) and converts all primed RNA within a sample to cDNA, regardless of its abundance, length or secondary structure. The most characterized RTs used for molecular biology are the retroviral RTs: avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV or MuLV) ). Genetic engineering and development of proprietary RT-enhancing buffers have led to the commercial availability of new enzymes that offer superior performance over these naturally occurring RTs. AMV and M-MLV reverse transcriptases are generally used to produce a DNA copy of the RNA template using either random primers, an oligo(dT) primer or sequence-specific primers. Some thermostable DNA polymerases (e.g., Tth DNA polymerase) possess a reverse transcriptase activity, which can be activated by using manganese instead of magnesium as a cofactor (Myers and Gelfand, 1991). After this initial reverse transcription step to produce the cDNA template, basic PCR is carried out to amplify the target sequence. The quality and purity of the RNA template is crucial to the success of RT-PCR. Total RNA or poly(A)+ RNA can be used as the starting template, but both must be intact and free of contaminating genomic DNA. Specific capture of poly(A)+ RNA will enrich a targeted message so that less of the reverse transcription reaction is needed for subsequent amplification. The efficiency of the first-strand synthesis reaction, which can be related to the RNA quality, also will significantly affect amplification results. GoScript™ Reverse Transcriptase is a formulation of M-MLV reverse transcriptase and optimized buffers designed for efficient and reproducible synthesis of first-strand cDNA from a full range of rare and abundant transcripts, even with difficult templates and in the presence of PCR inhibitors. GoScript is qualified for use in qPCR and is compatible with GoTaq® RT-qPCR Systems. GoScript is available in convenient mixes with either Oligo(dT) primers or random primers, as part of a complete kit, and as a stand-alone enzyme.RT PCR Products and Resources GoScript® Reverse Transcriptase is available as a standalone reverse transcriptase or as one-step and two-step RT-PCR kits. View ProductSee all RT-PCR Products Hot-Start PCR Hot-start PCR is a common technique to reduce nonspecific amplification due to assembly of amplification reactions at room temperature. At room temperature, PCR primers can anneal to template sequences that are not perfectly complementary. Since thermostable DNA polymerases have activity at these low temperatures (although in most cases the activity is less than 25%) the polymerase can extend misannealed primers. This newly synthesized region then acts as a template for primer extension and synthesis of undesired amplification products. However, if the reaction is heated to temperatures >60°C before polymerization begins, the stringency of primer annealing is increased, and synthesis of undesired PCR products is avoided or reduced. Hot-start PCR also can reduce the amount of primer-dimer synthesized by increasing the stringency of primer annealing. At lower temperatures, PCR primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer dimer, which often appears as a diffuse band of approximately 50–100bp on an ethidium bromide-stained gel. The formation of nonspecific products and primer-dimer can compete for reagent availability with amplification of the desired product. Thus, hot-start PCR can improve the yield of specific PCR products. To perform manual hot-start PCR, reactions are assembled on ice or at room temperature, but one critical component is omitted until the reaction is heated to 60–65°C, at which point the missing reagent is added. This omission prevents the polymerase from extending primers until the critical component is added at the higher temperature where primer annealing is more stringent. However, this method is tedious and increases the risk of contamination. A second, less labor-intensive approach involves the reversible inactivation or physical separation of one or more critical components in the reaction. For example, the magnesium or DNA polymerase can be sequestered in a wax bead, which melts as the reaction is heated to 94°C during the denaturation step, releasing the component only at higher temperatures (Carothers et al. 1989; Krishnan et al. 1991; Clark, 1988). The DNA polymerase also can be kept in an inactive state by binding to an oligonucleotide, also known as an aptamer (Lin and Jayasena, 1997; Dang and Jayasena, 1996) or an antibody (Scalice et al. 1994; Sharkey et al. 1994). This bond is disrupted at the higher temperatures, releasing the functional DNA polymerase. Finally, the DNA polymerase can be maintained in an inactive state through chemical modification (Moretti, T. et al 1998). Hot-Start PCR Products and Resources GoTaq® G2 Hot Start Taq is available as a standalone enzyme or master mix, In this formulation, the Taq polymerase is bound to a proprietary antibody that blocks activity. Activity is restored during initial denaturation, allowing hot-start PCR. View Hot Start PolymeraseView Master MIxes Long-Range PCR Amplification of long DNA fragments is desirable for numerous applications such as physical mapping applications (Rose, 1991) and direct cloning from genomes. While basic PCR works well when smaller fragments are amplified, amplification efficiency (and therefore the yield of amplified fragments) decreases significantly as the amplicon size increases over 5kb. This decrease in yield can be attributed to the accumulation of truncated products, which are not suitable substrates for additional cycles of amplification. These products appear as smeared, as opposed to discrete, bands on a gel. In 1994, Wayne Barnes (Barnes, 1994) and other researchers (Cheng et al. 1994) examined factors affecting polymerization across larger regions of DNA by thermostable DNA polymerases and identified key variables affecting the yield of longer PCR fragments. They devised an approach using a mixture of two thermostable polymerases to synthesize longer PCR products. The first polymerase lacks a 3′→5′ exonuclease (proofreading) activity; the second enzyme, present at a reduced concentration, contains a potent proofreading activity. Presumably, when the nonproofreading DNA polymerase (e.g., Taq DNA polymerase) misincorporates a dNTP, subsequent extension of the newly synthesized DNA either proceeds very slowly or stops completely. The proofreading polymerase (e.g., Pfu DNA polymerase or Tli DNA polymerase) serves to remove the misincorporated nucleotide, allowing the DNA polymerases to continue extension of the new strand. Although the use of two thermostable DNA polymerases can significantly increase yield, other conditions can have a significant impact on the yield of longer PCR products (Cheng et al. 1995). Logically, longer extension times can increase the yield of longer PCR products because fewer partial products are synthesized. Extension times depend on the length of the target; times of 10–20 minutes are common. In addition, template quality is crucial. Depurination of the template, which is promoted at elevated temperatures and lower pH, will result in more partial products and decreased overall yield. In long PCR, denaturation time is reduced to 2–10 seconds to decrease depurination of the template. Additives, such as glycerol and dimethyl sulfoxide (DMSO), also help lower the strand-separation and primer-annealing temperatures, alleviating some of the depurination effects of high temperatures. Cheng et al. also found that reducing potassium concentrations by 10–40% increased the amplification efficiency of longer products (Cheng et al. 1995). Long PCR Master Mix GoTaq® Long PCR Master MIx contains hot start Taq in a specially formulated mixture with a proprietary thermal stable proofreading polymerase. This optimized enzyme mixture allows efficient amplification of up to 40kb from lambda DNA or 30kb from human genomic DNA. View Long PCR Master Mix.