Maximizing PCR and RT-PCR Success

16 PCR Brochure 10/2016 Multiplex PCR Multiplex PCR employs different primer pairs in the same reaction for simultaneous amplification of multiple targets. This type of PCR often requires extensive optimization of annealing conditions for maximum amplification efficiency of the different primer–template systems and is often compromised by nonspecific PCR artifacts. A stringent hot-start procedure and specially optimized buffer systems are absolutely crucial for successful multiplex PCR. In our Multiplex PCR User Guide we provide tips and tricks to overcome challenges in multiplex PCR (Figure 17). Compared with standard PCR systems using only two primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products. This often results in unamplified DNA sequences and absence of expected PCR products. QIAGEN has overcome this problem with a specially adapted multiplex PCR buffer (provided with QIAGEN Multiplex PCR Kit and the QIAGEN Multiplex PCR Plus Kit). This buffer contains a unique synthetic additive, Factor MP, which further promotes stable and efficient annealing of different primers to the nucleic acid template. The increased hybridization efficiency and primer stability provides excellent product yields – even for primer pairs that normally bind suboptimally to their target sequence under the chosen conditions (Figure 18). This principle has been extended with the Type-it® Microsatellite PCR Kit, where each step of the procedure has been preoptimized – from template amount to precise cycle number. With this unique kit format, development of microsatellite assays with a high number of targets is easier than ever before (Figure 19). Table 4. Increasing success with challenging PCR applications Application Challenge Amplification method Genotyping Parallel amplification of multiple products Multiplex PCR High-throughput/fast PCR Reduce time to result Multiplex PCR/fast-cycling, hot-start PCR Single-cell PCR Increased sensitivity Highly specific hot-start PCR Cloning Sequence accuracy Amplification of long PCR products High-fidelity PCR Long-range PCR Detection of methylated DNA PCR specificity Methylation-specific PCR Viral load monitoring and gene expression analysis Increased sensitivity One-step RT-PCR Figure 17. Multiplex PCR Webapp.

Pages Overview