Maximizing PCR and RT-PCR Success

18 PCR Brochure 10/2016 Fast-cycling PCR Faster PCR amplification enables increased PCR throughput and allows researchers to spend more time on downstream analysis. The demand for reducing time-to-result (see Figure 1, page 2) is met by the recent development of faster PCR techniques. Fast PCR can be achieved using new thermal cyclers with faster ramping times or through innovative PCR chemistries that allow reduced cycling times due to significantly shortened DNA denaturation, primer annealing, and DNA extension times. Fast-cycling PCR reagents must be highly optimized to ensure amplification specificity and sensitivity. The QIAGEN Fast Cycling PCR Kit enables successful fast- cycling, hot-start PCR even on standard thermal cyclers, through the use of the novel Q-Bond® Molecule. This molecule dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, allowing the annealing time to be reduced to just 5 seconds. The unique buffer formulation and optimized DNA polymerase concentration also enables a significant reduction in denaturation and extension times. Visit www.qiagen.com/ PCR-literature to access literature on fast-cycling PCR. Single-cell PCR Single-cell PCR provides a valuable tool for genetic characterization using a limited amount of starting material. By flow cytometry or micromanipulation, individual cells of interest can be isolated based on cell-surface markers or physical appearance. Amplification of low-abundance template molecules – as low as one or two gene copies – requires a PCR system that is highly efficient, specific, and sensitive, such as HotStarTaq Plus DNA Polymerase (Figure 20). Long-range PCR PCR products of up to 4 kb can be routinely amplified using standard PCR protocols. However, amplification of PCR products longer than 4 kb often fails without lengthy optimization. Reasons for failure include nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions – all factors which have a greater effect on the amplification of longer PCR products than on shorter ones. “ Your kit allowed me to consistently and reproducibly amplify forensic DNA samples from 5000 year old bone samples in a short amount of time. This is a great tool for obtaining clean results in the shortest amount of time possible Dr. Alex Nikitin. Assistant Professor, Grand Valley State University, Minnesota. H o t - s t a r t e n z y m e ( S u p p l i e r T I ) H o t S t a r T a q P l u s ( Q I A G E N ) M – 500 bp Figure 20. Successful single-cell PCR. A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq Plus DNA Polymerase (QIAGEN) and a hot-start enzyme and buffer from Supplier TI (Hot-start enzyme). M: marker. A single specific fragment was only attained using HotStarTaq Plus DNA Polymerase. “

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