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#DESIGNING TOUCHDOWN PCR SOFTWARE#
#DESIGNING TOUCHDOWN PCR FREE#

Several advances in PCR have dramatically reduced the duration of PCR amplification reactions. Yellow box indicates optimal temperatures. Arrows indicate the annealing temperature that provided the highest specificity while maintaining good yield. Four different primer sets (A, B, C, and D) were designed and tested for amplification. All reactions were evaluated in a single run. Consult the PCR Assay Troubleshooting page for more help. If satisfactory results are not obtained at any annealing temperature, additional optimization steps must be taken, and primer redesign may be necessary. A sample annealing temperature optimization experiment is shown in Figure 1. The optimal annealing temperature is the one that results in the highest yield with no nonspecific amplification. If nonspecific amplification has occurred, additional bands will appear on the gel. Analyze the results using agarose gel electrophoresis. To find the optimal annealing temperature for your reaction, test a range of temperatures above and below the calculated T m of the primers. The gradient feature allows you to test a range of temperatures simultaneously, optimizing the annealing temperature in a single experiment. All Bio-Rad thermal cyclers offer a gradient feature. The optimal annealing temperature for an assay can be easily determined using PCR instruments that have a thermal gradient feature. Similar time-consuming tests may also be required to optimize the denaturation temperature. This involves repeating a reaction at many different temperatures. Even after calculating the T m of a primer, you may need to determine the annealing temperature empirically. On the other hand, setting the annealing temperature too high may reduce the yield of a desired PCR product. Setting the annealing temperature too low may lead to amplification of nonspecific PCR products. Optimizing the annealing temperature of your PCR assay is one of the most critical parameters for reaction specificity.

Verify specificity using tools such as the ( Basic Local Alignment Search Tool BLAST).

Check the sequence of forward and reverse primers to ensure no 3' complementarity (avoid primer-dimer formation).

Avoid repeats of Gs or Cs longer than 3 bases.

Avoid secondary structure adjust primer locations so that they are located outside secondary structure in the target sequence, if required.

One way to calculate T m values is by using the nearest-neighbor method (use this online T m calculator)

Design primers that have a GC content of 50–60%.

When designing primers for a PCR assay, follow these guidelines:

Choose a region that has a GC content of 50–60%.

Avoid regions with long (>4) repeats of single bases.

See Real-Time PCR: General Considerations (Bio-Rad bulletin 2593) for more details Use programs such as mfold to predict whether a PCR product will form any secondary structure at the annealing temperature.

When possible, avoid regions that have secondary structure.

Although short PCR products are typically amplified with higher efficiency than longer ones, a PCR product should be at least 75 bp to easily distinguish it from any primer-dimers that could potentially form See the guidelines under Long PCR Assays when amplifying long sequences. When amplifying any sequence in a given section of DNA for purposes such as genotyping experiments, follow these guidelines to select a product.

#DESIGNING TOUCHDOWN PCR SOFTWARE#

Commercially available programs such as Beacon Designer software can perform both primer design and target sequence selection.

#DESIGNING TOUCHDOWN PCR FREE#

Assess primer properties (melting temperature, secondary structure, complementarity).Ī number of free online resources are available to help you with PCR assay design (see Free Internet Resources for Primer Design).Check the literature and databases for existing primers.When designing primers for a PCR assay, follow these steps:

The use of PCR primers specifically designed and validated for PCR assays with your target of interest is highly recommended. Therefore, care must be taken when choosing a target sequence and designing primers. Both the primers and the target sequence can affect the efficiency, specificity, and accuracy of PCR assays. A successful PCR assay requires efficient and specific amplification of the product.