Challenges in Amplifying GC-rich DNA
The polymerase chain reaction (PCR) technique for DNA amplification is a vital tool in medical development and biological research, such as infectious disease diagnostics, molecular genetic analysis, and the creation of recombinant DNA constructs for protein expression. While PCR technology is generally straightforward, amplifying DNA sequences with high guanine-cytosine (GC) content presents specific challenges. GC-rich sequences are prevalent in crucial regulatory elements of human DNA, such as promoters, enhancers, certain essential housekeeping genes, tumor suppressor genes, and tissue-specific genes. Some bacterial genomes characterized by high GC content, such as Streptomyces, Mycobacterium, and Pseudomonas.
The challenges in amplifying GC-rich DNA primarily are high stability secondary structures, annealing temperature optimization, and polymerase stalling. The formation of three hydrogen bonds between guanine (G) and cytosine (C) leads to the propensity of GC-rich DNA regions to form highly stable secondary structures such as stem-loop structures (also called hairpins). These stable structures are resistant to denaturation during the PCR annealing and extension phases, hindering effective primer binding to the template and potentially causing premature termination of polymerase extension, thereby reducing amplification efficiency or even leading to amplification failure. High annealing temperatures are required to achieve effective denaturation of these secondary structures, but excessively high annealing temperatures may affect the binding of primers to templates, reducing the PCR amplification efficiency. Therefore, the window of annealing temperatures that effectively denatures the GC-rich DNA templates while ensuring primer binding to the template is often narrow. In addition, DNA polymerases tend to stall when encountering high GC sequences, resulting in incomplete extension products.
Strategies employed in PCR optimization to address these challenges include:
- Optimization of PCR buffer composition: Some additives can reduce DNA duplex stability by interfering with hydrogen bonds between bases, such as dimethyl sulfoxide (DMSO), betaine, and formamide. Additionally, adjusting the magnesium ion (Mg2+) concentration in the buffer can enhance polymerase activity.
- Optimization of primer design strategies: During primer design, the GC content of primers is appropriately reduced, and the accumulation of GC bases at the primer termini is minimized. Furthermore, advanced primer design software offers algorithms specifically tailored for GC-rich templates to aid in predicting and avoiding the formation of stable secondary structures.
- Adjustment of cycling parameters: For templates with high GC content, higher annealing temperatures and longer extension times are required to ensure sufficient primer binding to the template and complete extension of the entire target fragment by the polymerase.
- Hot-start PCR technology: Hot-start PCR effectively suppresses non-specific amplification and is ideal for the amplification of GC-rich templates, which can improve the yield and specificity of the target fragment.
- Selection of high-performance DNA polymerases: Polymerases with enhanced strand displacement activity and high thermostability exhibit improved performance in the amplification of high GC content templates.
GC-Rich DNA Target Kit and GC TEMPase Master Mixes
Amerigo Scientific offers a unique solution for amplification of GC-rich and other difficult DNA targets. The GC-rich DNA Target Kit includes TEMPase Hot Start DNA Polymerase with two special buffers and extra MgCl2 (25 mM), which are optimized to amplify GC-rich DNA targets that cannot be amplified by conventional master mixtures. We also provide GC Buffer I or GC Buffer II, used in combination with TEMPase Hot Start DNA polymerase, for promoting excellent amplification of DNA targets with varying high degrees of GC content.
When failure is encountered in PCR using the conventional TEMPase Hot Start Master Mix and buffer, please try GC buffer I as the master mix or a kit for better results. If the amplification is still not satisfactory, then switch to GC buffer II.
Features
All these products are manufactured in-house in accordance to our ISO 9001:2015 quality management system, ensuring that each batch possess the same robust performance.
- High success rate of GC-rich DNA amplification
- High specificity, sensitivity, and product yield
- Diminished formation of non-specific product
- Reaction set-up at room temperature
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