Maximizing PCR and RT-PCR Success

8 PCR Brochure 10/2016 Magnesium ion concentration Magnesium ions are a critical DNA polymerase cofactor necessary for enzyme activity. In a manner similar to K+ (see Figure 7), Mg2+ also binds to DNA, primers and nucleotides contained in the amplification reaction. The Mg2+ concentration is generally higher than that of dNTPs and primers, and some optimization may be necessary for different template and primer concentrations. Higher than optimal concentrations of Mg2+ can stabilize nonspecific binding and is often indicated by decreased yields of specific PCR products (Figure 9) and the appearance of background smear or other PCR artifacts. The destabilizing effect of NH4 + (provided in the QIAGEN PCR Buffer) on nonspecific primer annealing maintains the predominance of specific annealing over a range of Mg2+ concentrations and greatly reduces the need to optimize Mg2+ concentration. M 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Supplier TI 45 65°C 45 Supplier TI 65°C ! M 45 65°C 1 2 3 4 5 6 7 1 2 3 4 5 6 7 45 QIAGEN 65°C ! Supplier D Figure 8. Influence of annealing temperature on one-step RT-PCR specificity. One-step RT-PCR was performed using kits from the indicated suppliers (two kits from supplier TI ) over a range of annealing temperatures. A 1289 bp fragment from the human RCC1 gene was reverse transcribed and amplified from Hela RNA (arrow). M: marker. High levels of specific amplification without optimization were observed only with the QIAGEN OneStep RT-PCR Kit. Figure 9. Influence of Mg2+ concentration on PCR success. A 750 bp product from the human PRP gene was amplified in 50 μl reactions using 20 ng genomic DNA from leukocytes and DNA polymerases from the indicated suppliers. PCR buffer contained 1.5, 2.5 or 3.5 mM Mg2+ . PCR products (5 μl) were subjected to electrophoresis on a 1.5% agarose gel. M: marker. High levels of specific amplification at all Mg2+ concentrations were observed only using QIAGEN‘s Taq DNA Polymerase and its innovative buffer system. M M mM Mg2+ 1.5 2.5 3.5 QIAGEN 1.5 2.5 3.5 Supplier BIV 1.5 2.5 3.5 Supplier TI – 750 bp Annealing temperature The optimal primer annealing temperature is dependent on the base composition (i.e., the proportion of A, T, G, and C nucleotides), primer concentration, and ionic reaction environment. Using QIAGEN PCR buffers, containing both K+ and NH4 + , delivers high yields of specific PCR product over a wide range of annealing temperatures. This specificity is achieved by destabilizing nonspecifically bound primers, providing a more robust reaction environment and eliminating the need for tedious annealing temperature optimization. In contrast, the range of optimal PCR annealing temperatures is smaller and less predictable when using a PCR or one-step RT-PCR buffer that only contains K+ , as illustrated in Figure 8. M 12345671234567 4565°C 45 4565°C 12345671234567 1.52.53.5 1.52.53.5 1.52.53.5

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