Overcoming Challenges in End-Point PCR

Learn how QIAGEN’s PCR technologies enable success at the first attempt
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PCR is a widely used technique in many research areas such as biomarker discovery, gene regulation, and cancer research. The demand for increased throughput, reduced costs, higher assay sensitivity, and reliable data normalization has increased significantly. Despite recent improvements in PCR technologies, researchers still face challenges such as nonspecific amplification, low yields, and smearing, especially when dealing with difficult templates (with a high GC content) or low template amounts. This leads to an increased need for optimization of PCR parameters, such as cycling conditions, and Mg2+ and dNTP concentrations.

QIAGEN’s extensive PCR portfolio offers dedicated kits and solutions to meet all your requirements. All QIAGEN PCR kits consist of the unique QIAGEN PCR Buffer and highly stringent PCR enzymes, both of which eliminate the need for optimization of PCR parameters. In addition, our PCR kits have been functionally validated with the QIAxcel System for effortless fragment analysis, providing the best possible combination for PCR and fragment detection.

This article addresses how QIAGEN’s PCR technologies help overcome challenges associated with PCR and enable success at the first attempt. It also presents the advantages of using the QIAxcel for superior DNA fragment analysis in as little as 10 minutes.
Critical factors influencing PCR success
Effortless DNA fragment analysis
Critical factors influencing PCR success
Despite the fact that PCR amplification is performed routinely and that thousands of PCR protocols have been developed, researchers still encounter technical difficulties with PCR experiments and often fail to obtain specific amplification products. Although there are several different technical difficulties (e.g., smearing, low yield, and unspecific amplification), there are only two main reasons for PCR failure or poor results. These are the specificity of the reaction and template secondary structure.

The factors that influence PCR success can be broken down into:
  • Choice of enzyme
  • Reaction conditions (i.e., choice of buffer, annealing temperature, etc)
  • Primer design
  • Template quality
  • Application type
Choice of enzyme
Taq DNA polymerase is the most commonly used enzyme for standard end-point PCR. The robustness of this enzyme allows its use in many different PCR assays. However, as this enzyme is active at room temperature, it is necessary to perform reaction setup on ice to avoid unspecific amplification. QIAGEN has overcome this limitation with the introduction of the novel TopTaq DNA Polymerase. This innovative non-hot-start enzyme can be stored routinely in the fridge at 4°C and is stable at room temperature allowing immediate reaction setup without the use of ice. For more challenging PCR applications, the use of hot-start PCR is crucial for successful results. The unique hot-start procedure, based on chemical modification, provided with QIAGEN hot-start enzymes is easily incorporated into any PCR program using a simple 5 (HotStarTaq Plus DNA Polymerase) or 15 minute (HotStarTaq DNA Polymerase) initial enzyme activation step. For applications requiring a low error rate, such as cloning and site-directed mutagenesis QIAGEN has developed HotStar HiFidelity DNA Polymerase. This high-fidelity polymerase incorporates a hot-start activation to its exonuclease activity, giving reliable and sensitive results. In addition, this enzyme also adds an A overhang during the final extension step allowing direct UA- or TA-cloning.
Reaction conditions
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. QIAGEN has found that the balanced combination of K+ and NH4+ used in all QIAGEN PCR Buffer formulations can strongly increase primer annealing specificity over a broad range of annealing temperatures. The improved specificity is caused by ammonium ions destabilizing the weak hydrogen bonds at mismatched bases. The use of these buffers also often eliminates the need for optimization of Mg2+ concentration or annealing temperature for different primer-template systems and maintains high primer annealing specificity in each PCR cycle.
Primer design
Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR. Because PCR consists of multiple rounds of enzymatic reactions, it is more sensitive than single-step enzyme-catalyzed processes to impurities, such as proteins, phenol, chloroform, salts, ethanol, EDTA, and other chemical solvents. We recommend QIAGEN sample prep technologies using membrane-based kits (e.g., QIAamp and QIAprep Kits) for template preparation to ensure reliable PCR results.
Application type
QIAGEN offers a wide variety of PCR kits that have been specifically developed to provide accurate and reliable results in a variety of applications. These include genotyping, cloning, single-cell PCR, high-throughput/fast PCR, methylated DNA detection, viral load monitoring, and many more. Our range of products offer convenient features such as master mix formats, gel loading PCR buffers, and streamlined protocols highly suited for detection platforms such as automated electrophoresis systems like the QIAxcel System (see figure QIAxcel Advanced System). In addition, our kits contain special additives that dramatically improve PCR performance. Most QIAGEN PCR kits include the novel reagent Q-Solution, which changes the dynamics of the PCR reaction to increase sensitivity when amplifying GC-rich DNA and targets with strong secondary structures (see figure Multiplex PCR using Q-Solution). Our innovative PCR buffer formulations contain a balanced combination of K+ and NH4+ which strongly increase primer annealing specificity (see figure NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing).

We have dedicated kits for a range of amplification methods such as fast-cycling, hot-start, multiplex, high-fidelity, and long-range PCR, ensuring greater flexibility. Visit our online selection guide to choose the kit best suited to your needs. 

Effortless DNA fragment analysis
Currently, the most commonly used method for nucleic acid separation is gel electrophoresis, using manually poured slab gels. However, this method is highly labour-intensive and exposes users to hazardous chemicals such as ethidium bromide. In addition, thorough analysis of the data in terms of fragment sizes and concentration can be tedious especially when the data is to be compared with previously analyzed PCR products. Standardization is of key importance when comparing data from different gel runs and this places a great emphasis on accurate electrophoresis conditions and record keeping.

Analysis of end-point PCR products can be made easier using the QIAxcel Advanced System, which fully automates high-resolution capillary electrophoresis of up to 96 samples without manual intervention (see figure QIAxcel Advanced System). It also ensures greater safety as exposure to hazardous chemicals such as ethidium bromide is minimized. The high detection sensitivity provided by the QIAxcel analyzer enables robust results even with low concentrations of nucleic acid (see figure High resolution analysis of amplicons using the QIAxcel). Standardization is achieved through the use of pre-cast gel cartridges that allow up to 96 samples to be run using exact electrophoresis conditions. Results can be viewed in real-time and the data can be easily stored and analyzed allowing simple comparison of data generated from different sample runs. Therefore, the QIAxcel system speeds up nucleic acid analysis applications by eliminating slab-gel analysis while streamlining entire sample purification and analysis workflow.

Our extensive range of PCR kits, in combination with the QIAxcel, enable fast and convenient analysis and ensure increased reproducibility and reliability.

For more detailed information about PCR challenges and solutions, see the Maximizing PCR and RT-PCR success — Second Edition brochure. For more information about the QIAxcel, see the QIAxcel — Pure Excellence brochure.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.


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