Maximizing PCR and RT-PCR Success

PCR Brochure 10/2016 19 Preventing DNA damage, such as DNA depurination, is of particular importance for amplification of long PCR products, as a single DNA lesion within the template is sufficient to stall the PCR enzyme. DNA damage during PCR cycling can be minimized with specific buffering substances that stabilize the pH of the reaction. The QIAGEN LongRange PCR Kit is optimized for the amplification of PCR products up to 40 kb in size. DNA pre-incubated in QIAGEN LongRange PCR Buffer shows similar PCR product yield compared to non-damaged control DNA, demonstrating that the protecting function of the buffer system provides an optimal reaction environment for the amplification of long PCR products (Figure 21). Unlike kits from other suppliers, the QIAGEN LongRange PCR Kit overcomes long-range PCR challenges, owing to its unique features (Figure 22). Figure 21. LongRange PCR Buffer protects genomic DNA from excessive damage. Genomic DNA from leukocytes in water (Water), long-range PCR buffer from Supplier A (Standard PCR buffer) or QIAGEN LongRange PCR Buffer (QIAGEN LongRange PCR Buffer) were used directly for PCR (–) or incubated at 95°C (+) before PCR. Amplification reactions (50 μl) were performed using the QIAGEN LongRange PCR Kit. M: marker. Greater DNA damage, as indicted by reduced product yield, is observed with pre-treatment of DNA in water or standard long-range PCR buffers than with pre-treatment in QIAGEN LongRange PCR Buffer. Figure 22. Successful long-range PCR using the QIAGEN LongRange PCR Kit. PCR was performed with the indicated kits, using 40 ng human genomic DNA as a template. In contrast to kits from Supplier R and Supplier T, the QIAGEN LongRange PCR Kit provided successful amplification, resulting in amplicons of 7.6 kb and 8.9 kb, using a simplified, time-saving protocol. M: marker. M – – + + + + + + Water Standard PCR buffer QIAGEN LongRange PCR Buffer – 10 kb 95°C step A Supplier R Supplier T QIAGEN B A A B B M Methylation-specific PCR (MSP) MSP enables the methylation status of target DNA to be determined after sodium bisulfite treatment (e.g., using the EpiTect® Fast Bisulfite Kit). The method requires two sets of primers to be designed: one set that anneals to unchanged cytosines (i.e., methylated in the genomic DNA) and one set that anneals to uracil resulting from bisulfite treatment of cytosines not methlyated in the genomic DNA. Amplification products derived from the primer set for unchanged sequences indicates the cytosines were methylated and thus protected from alteration.* Stringent and highly specific PCR conditions must be used to avoid nonspecific primer binding and the amplification of PCR artifacts. This is particularly important as the conversion of unmethylated cytosines to uracils reduces the complexity of the DNA and increases the likelihood of nonspecific primer–template binding. HotStarTaq Plus DNA Polymerase, designed for amplification of templates with high GC-content DNA, has been successfully used for MSP (Figure 23). In addition, QIAGEN’s dedicated EpiTect MSP Kit is specially optimized for highly reliable MSP in epigenetics applications. * Review article: Hernández H. G., et al. (2013) Optimizing methodologies for PCR-based DNA methylation analysis. Biotechniques 55, 181.

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