The PyroMark PCR Kit is specifically optimized for Pyrosequencing analysis, providing highly specific and unbiased amplification of template DNA for various applications, such as mutation detection, SNP analysis, methylation analysis, and sequencing. The kit is provided in a convenient master mix format consisting of HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer for highly specific amplification. In addition, Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is provided. The PyroMark PCR Kit is also supplied with CoralLoad Concentrate, which is strongly recommended for use with the PyroMark PCR Master Mix, for highly specific PCR and high yields of amplified DNA. CoralLoad Concentrate contains 2 gel-tracking dyes, allowing PCR products to be loaded directly on a gel and checked prior to Pyrosequencing.
The PyroMark PCR Kit enables PCR amplification of template DNA optimized for Pyrosequencing analysis. The convenient master mix format enables specific amplification of bisulfite converted DNA or genomic DNA from various starting materials, using only one protocol. Bisulfite treatment of genomic DNA converts nonmethylated cytosines to uracils and produces DNA that mainly consists of three bases. This less complex DNA is a challenging PCR template — the PCR products obtained are likely to have low yields, and nonspecific products might also be generated due to increased probability for mispriming. The balanced combination of salts in the PyroMark PCR Kit's unique master mix ensures specifically targeted primer binding and prevents mispriming. The special formulation also prevents accumulation of excess biotinylated primer and minimizes generation of artifacts that can interfere with the Pyrosequencing reaction. The high yields of specific PCR products obtained ensure reliable Pyrosequencing results.
During the annealing step of every PCR cycle, the buffer composition ensures a high ratio of specific-to-nonspecific primer binding (see figure " Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations, compared with conventional PCR buffers. Optimizing PCR by varying the annealing temperature or Mg2+ concentration is usually not required.
HotStarTaq DNA Polymerase, provided in the master mix, is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer–dimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15 minute, 95°C incubation step, that can easily be incorporated into existing thermal cycling programs. HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product. PCR setup is quick and convenient, since all reaction components can be combined at room temperature.
The PyroMark PCR Master Mix is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent often enables or improves PCR caused by difficult templates (e.g., templates with a high degree of secondary structure or templates that are GC-rich). Unlike other commonly used PCR additives, such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. PCR fragments amplified in the presence of Q-Solution can be successfully analyzed by Pyrosequencing.
The kit is also supplied with CoralLoad Concentrate, which contains a gel loading reagent and 2 gel tracking dyes (see figure " CoralLoad Concentrate"). When using CoralLoad Concentrate, the PCR products can be directly loaded onto an agarose gel without prior addition of loading buffer. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate can be successfully analyzed by Pyrosequencing.
The reaction composition and cycling conditions are optimized for amplifying template DNA for Pyrosequencing analysis. Template DNA is combined with the convenient PyroMark PCR Mastermix, along with Q-Solution, CoralLoad concentrate, and amplification primers (one of which is biotinylated). HotStarTaq DNA Polymerase is inactive at room temperature, so it is not necessary to keep the reactions on ice during setup.