Maximizing PCR and RT-PCR Success - Reaction conditions

PCR Brochure 10/2016 7 Reaction conditions Primer annealing specificity and PCR buffers In PCR, annealing occurs between the primers and complementary DNA sequences in the template. Primer annealing must be specific for successful amplification. Due to the high concentration of primers necessary for efficient hybridization during short annealing times, primers can anneal to non-complementary sequences. Amplification of products from nonspecific annealing competes with specific amplification and may drastically reduce the yield of the specific product (Figure 2, page 4). The success of PCR largely depends on maintaining a high ratio of specific to nonspecific annealing of the primer molecules. Annealing is primarily influenced by the components of the PCR buffer (in particular the cations) and annealing temperature. Special cation combinations can maintain high primer annealing specificity over a broad range of annealing temperatures. This eliminates the need for optimization of annealing temperatures for each individual primer–template system and also allows the use of non-ideal PCR systems with different primer annealing temperatures. A balanced combination of cations promotes specific primer annealing Cations in commonly used PCR buffers bind to the negatively charged phosphate groups on the DNA backbone and thereby neutralize these negative charges. This weakens the electrorepulsive forces between the DNA template and primer molecule leading to more stable hybridization of the primer. Most commercially available PCR buffers contain only one monovalent cation, K+ , which stabilizes both specific and nonspecific primer annealing. This often results in smearing and nonspecific DNA amplification, which leads to lower product yields. QIAGEN has found that the balanced combination of K+ and NH4 + used in all QIAGEN PCR buffer formulations can strongly increase primer annealing specificity. The improved specificity is caused by ammonium ions destabilizing the weak hydrogen bonds at mismatched bases (Figures 6 and 7). H K+ P– B Primer Template Destabilization Stabilization NH4 + NH3 + H + B P– K+ Figure 6. Animation on QIAGEN‘s unique PCR buffer system. Watch the video on www.qiagen.com/resources/e-learning/videos/pcr-buffer/. Figure 7. Effect of unique QIAGEN PCR Buffer. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4 + , which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to non-specific primer–template binding over a wide temperature range.

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