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Nucleic Acid Amp Profiling

Taq PCRx® DNA Polymerase and PCRx Enhancer System

Taq PCRx DNA Polymerase,is supplied with an optimized buffer system including 10X PCRx Amplification Buffer, 50 mM Magnesium Sulfate, and 10X PCRx Enhancer Solution, in addition to 50 mM Magnesium Chloride and the standard 10X PCR Buffer. PCRx Enhancer Solution is the cosolvent that simplifies Polymerase Chain Reaction (PCR) amplification of problematic and/or GC-rich templates using standard dNTPs and thermocycling protocols. PCRx enhancer has also been shown to be helpful in Taq-based sequncing reactions of difficult sequences. This optimized buffer system and PCRx enhancer can also be purchased separately as the PCRx Enhancer System.

PCR optimization (1) is often a difficult and time consuming process requiring adjustment of incubation times and temperature, magnesium, primer, dNTP, Taq DNA polymerase and DNA template concentration, and potentially, the design of multiple primer sets. DNA sequences containing stable secondary structure(s) are frequently refractory to conventional PCR optimization strategies as these sequences are resistant to denaturation and pose barriers to primer annealing or procession of DNA polymerase.

10X PCRx Enhancer Solution is a novel PCR cosolvent (patent pending) that facilitates efficient amplification of GC-rich sequences and remedies difficulties associated with PCR amplification of problematic templates. For problematic and/or GC-rich templates, the combination of 10X PCRx Enhancer Solution and 10X PCRx Amplification Buffer offers higher primer specificity, broader magnesium concentration optima, broader annealing temperature optima, and improved thermostabilization of recombinant Taq DNA polymerase.

Components

  • The Taq PCRx DNA Polymerase includes:
    1. Taq DNA Polymerase, Recombinant
    2. 10X PCRx Enhancer Solution
    3. 50 mM MgSO4
    4. 10X PCRx Amplification Buffer
    5. 50 mM MgCl2
    6. 10X PCR Reaction Buffer Minus Mg. (200 mM Tris pH 8.4, 500 mM KCl).

    The PCRx Enhancer System includes:
    1. 10X PCRx Amplification Buffer
    2. 50 mM MgSO4
    3. 10X PCRx Enhancer Solution (Cosolvent)

General Recommendations and Guidelines:

  • Buffer Selection
    Taq PCRx DNA polymerase is supplied with two different buffers to provide optimal reaction conditions for the widest variety of DNA templates. The standard 10X PCR Buffer and 50 mM MgCl2 are recommended for PCR of routine templates (30-50% GC content).

    Conventional PCR buffer components in established protocols can be directly substituted with the 10X PCRx Amplification Buffer and 50 mM MgSO4 resulting in more robust amplification and improved product yield in some cases. However, optimal reaction conditions vary and may need to be evaluated by the customer. Use of 10X PCRx Enhancer Solution for PCR of GC-rich templates results in wider reaction optima and significantly improves the probability of successful PCR amplification.

    note: Targets with GC content ++ concentrations (>/=2 mM) or use of the standard PCR buffer for optimal amplification. In addition, primer sets that generate specific product using standard PCR buffer may not benefit from using PCRx Amplification Buffer.

    PCRx Enhancer Solution
    Optimal concentration of 10X PCRx Enhancer Solution will vary depending on GC content, Mg++ concentration, and annealing temperature. Many GC-rich templates (up to 80%) may benefit by simply using the optimized 10X PCRx Amplification Buffer without added PCRx Enhancer solution. For targets with 45 to 60% GC, we recommend testing 10X PCRx Enhancer Solution at 0X, 0.5X, and 1X final concentration. Targets with higher GC content (65 to 90%), or simple repeat sequences, may require up to 4X concentration.

    10X PCRx Enhancer Solution lowers DNA melting temperature (Tm). Consequently, the maximum primer annealing temperature is lowered approximately 2°C per 1X PCRx Enhancer Solution concentration; however, effective annealing temperatures are widened over a much broader range. While no single thermal cycling protocol is optimal for every template, we recommend starting with an annealing temperature of 55°C to 60°C and varying the amount of 10X PCRx Enhancer Solution.

    Addition of 10X PCRx Enhancer Solution extends the range of effective Mg++ concentration (1 to 3 mM). For most target sequences, best results are obtained using 1.5 mM MgSO4.

    Best results with PCRx reagents have been obtained using 2.5 units of Taq DNA Polymerase in a 50-µl amplification reaction. For long templates (up to 10 kb) or six-fold higher fidelity, we recommend using PLATINUM® Taq DNA Polymerase High Fidelity (Cat. Nos. 11304-011/-029).

    Primer Design
    Effective primer design is the single most important parameter in PCR. PCR primers should be designed to: form stable, highly specific duplexes with the target sequence, minimize potential for secondary structure and dimer formation, minimize 3'-terminal complementarity (primer dimer), contain high stability (i.e., GC clamps) in the central and/or 5'-region, contain low stability in 3'-region (no more than two C or G in last five 3'-bases) to maximize specificity of priming, and have an equivalent/balanced Tm (DNA melting temperature).

    Primers for high GC content templates should have a GC content similar to that of the target sequence and a Tm of at least 70°C as calculated using nearest neighbor algorithms. For optimum PCR efficiency, the difference between PCR product Tm and primer Tm should be less than 25°C. Primer Tm can be adjusted by increasing primer length at the 5'-end. Ideally, primers should be 20 to 25 bases long; however, primers up to 30 bases are effective for amplification of GC-rich sequences.

    Sequencing notes:
    • Using the ABI Prism Dye Terminator Cycle Sequencing ready reaction kit, PCRx Enhancer does improve sequence quality when used at 2x, although the amount of enhancer used may need to be titrated.
    • PCRx Enhancer was effective in Original Dye Terminator cycle sequencing and in Big Dye cycle sequencing.
    • PCRx Enhancer is not recommended unless the template being sequenced is known to be problematic. Use of PCRx Enhancer may lead to a decrease in signal intensity, compared to no treatment at all.
    • PCRx buffer is not required. It is best to use the ABI buffer called for in the protocol.

Troubleshooting

  • PCRx Enhancer System Did Not Allow Generation of Amplification Products:
    • Make sure the template was not <45% GC. The Enhancer can inhibit the amplification of AT rich products.
    • Not enough Taq DNA Polymerase was added to the reaction. One should have at least 2.5 units in the reaction.
    • Make sure that the expected PCR product is not larger than 10 kb. PCRx did not tend to allow amplification of products >10 kb when tried using some primer sets for Ad2 DNA. Try titrating at concentrations up to 1X (e.g., 0.25X, 0.5X, 0.75X, 1.0X).
    • Make sure a lower annealing temperature is used for the primer than what would be calculated in the absence of PCRx. Remember that since PCRx Enhancer lowers the melting temperature of the DNA template, it will do the same for the primer/DNA complex also.

    NonSpecific Bands Remain/Specifity Not Improved
    • Annealing temperature and [Mg] may still need to be optimized.
    • Try the PCRx Enhancer solution at several different concentrations.
    • Make sure the PCRx Enhancer solution was used in the presence of the PCR Amplification Buffer and not just plain PCR buffer.
    • PCRx still is not a cure for bad primer design.

    PCR Products That Amplified in the Absence of PCRx Decrease In Yield In the Presence of PCRx.
    • If you are not having problems with the PCR, there is no need to use PCRx Enhancer Solution. If the target is GC content of 30-45%, use of PCRx reagents may result in lower PCR products. To enhance the yield of amplification of these products, perhaps use of PLATINUM Taq, High Fidelity may be the best choice.

    Downstream Reactions On PCR Products (Generated in the presence of PCRx) Do Not Work.
    • It is best to purify PCR products (ethanol precipitation, CONCERT PCR Purification System) to remove the PCR Enhancer reagent prior to subsequent reactions.

    PCR Enhancer Solution is Yellow
    • This is normal. We have seen this and it does not affect the performance of the product.