How do you design/find/order a primer for PCR?

Posted by Jack on December 9, 2022

Creating a PCR primer can be a complicated process and can take a long time to complete. But the end result is well worth the effort.

Placement of the 3' end of the primer

During the course of PCR, placement of the 3' end of the primer is of utmost importance. This is because the position of the primer affects its base pairing, which is important for a successful PCR. It also contributes to the amplification bias of the primer throughout the entire interaction with the template. The following guidelines will help you determine the best position for your primer.

The best position for a primer is at the 3' end, where the most specific and GC rich motifs are located. The location of these motifs will have a significant impact on amplification bias, particularly in family A DNA polymerases.

To get a better idea of the most optimal position for your primer, you can use a primer design program. These tools will guide you through the process and produce graphical output. Using the software, you can view an example of the target sequence along with the observed bias. You can download a csv file that will show you the optimal positions for your primer.

The most ideal melting temperature for a primer is between 60 and 64 degC, depending on the cycling conditions. This allows for the primer to bind at the same time. It is also necessary to ensure that the free energy of the primer is below -5 kcal/mol. This temperature allows the primer to bind to the template and amplify the product.

The best way to find the most optimal position for your primer is to use the iC-Architect software. This program will calculate the most optimal position for your primer based on your DNA polymerase preferences. You can run the program in your biology workbench. It will then create a csv file that will give you the most optimal positions for your primer. You can use this csv file to perform Tm calculations.

Another useful tool for finding the most optimal position for your primer is the Mfold box. This box will display the structure of the primer, as well as the 3' end of the primer. In addition, it will give you a downloadable csv file that you can use to perform Tm calculations.

Mismatched bases towards the middle of the primer

PCR is a process that amplification of genetic material from one strand of DNA to another. The process consists of three steps, each of which is temperature sensitive. First, the primer or probe must bind to a complementary region of single stranded DNA, then the enzyme extends the primer or probe, and finally, the product is amplified. These three steps are essential to a successful PCR experiment. However, a number of factors can influence the outcome of the experiment. For example, a mismatched base towards the middle of a primer for PCR can prevent amplification.

A single mismatch at the terminal 3' end of a primer for PCR can cause a decrease in amplification efficiency, which may lead to false-negative results. In addition, a single mismatch at the terminal 3' end prevents extension of the primer, which is essential for discriminating between different alleles. It can also lead to a two-fold underestimation of the initial copy number.

For diagnostic rPCR, the presence of a single mismatch at the 3' end of a primer is particularly problematic. In fact, it can completely prevent amplification and alter the cycle threshold. It is important to recognize that there are several ways to increase amplification efficiency.

For example, a mismatched G-A penultimate mismatch inhibits amplification more than a purine-purine mismatch. A purine-pyrimidine mismatch has a similar geometry to a G-C base pairing, which can be extended more efficiently by Taq polymerase. A third option is to introduce an additional mismatch at the terminal 3' end of the primer.

During mutagenesis screens, a primer for PCR should contain a mismatched base towards the middle of the primer. In addition, a primer should be designed with a balance between GC-rich and AT-rich domains. The distribution of these domains is important for stability of the primer. In addition, primers should avoid repeating bases.

The optimal melting temperature for primers is between 60-64degC. This temperature is determined based on the optimal temperature for PCR enzyme function and the cycling conditions. It is important to note that too low a melting temperature can result in non-specific products. A melting temperature of 62degC is typical, but higher temperatures are required to amplify products from polymorphic regions.


Whether you are designing a new primer, searching for a candidate PCR product, or checking the specificity of a pre-existing primer, Primer-BLAST is a useful tool. BLAST is a general-purpose target-specific PCR primer design tool that runs on a farm of machines at the NCBI.

Primer-BLAST uses a highly sensitive BLAST algorithm and a local alignment technique to design primers. You can submit either a template or a pre-existing primer as input, and the program will return a series of five specific primer pairs based on your specifications. You can specify the number of mismatches, the 3' end region, and a threshold for the number of mismatches.

The program also offers flexible options for stringent specificity check parameters. If you're designing a new primer, the average search time is 2.6 minutes, though this may vary depending on your selection of parameters.

The program also features a "fast manual primer creation" feature. If you're not satisfied with the results of the program, you can manually edit the primer's sequence and submit the result for review. The resulting primer will sit in the polymorphic region, which means you will have a better idea of the product you will be able to generate.

The program also provides the ability to select from a variety of target-specific primer pairs. This includes a list of five mRNA variants of the zinc finger protein 419 (ZNF419). It can also analyze transcript variants of other genes.

Primer-BLAST has a few tricks up its sleeve, including the possibility of detecting amplification targets with up to five mismatches. You can filter out targets with too many mismatches, and specify a nucleotide match threshold.

The program also has a number of other features, including an interactive mRNA product identifier, an mRNA variant database, and the ability to examine mRNA variants individually. It is also capable of searching the RefSeq mRNA database for a match to your target. In fact, it generated 11,236 hits from the RefSeq database.

The Primer-BLAST website is free and open to all users. The software can be downloaded and installed on a desktop or a server. It is designed to provide a level of flexibility and detection sensitivity that will help you get the most from your next PCR.

Prevent primer-dimer and hairpin formation

During PCR, homologous primers can form intermolecular interactions. This can decrease the amount of product produced and decrease yield. A new class of primers has been developed that reduces the impact of primer-dimer formation. This technology is based on the principle of cooperativity.

Cooperative primers are designed with a secondary structure that inhibits primer-dimer formation. They contain short sequences that bind to a target sequence to increase its concentration. They also have a low melting temperature. These characteristics make them ideal for amplification of a target DNA. They were originally developed for the beta-actin gene in the human mitochondrial sequence. In addition to preventing the formation of primer-dimers, they will allow efficient amplification of a template.

The primers were initially spiked with 150,000,000 primer-dimers. In order to determine their specificity, the amplification curves were analyzed. They were determined using real-time data, which was compared with the endpoint fluorescence signals. This analysis resulted in different endpoint Tp values.

The optimal annealing temperature for a primer pair is generally less than the melting temperature of the primer. Typically, the primers are melted at temperatures between 50 and 60 degC. When the annealing temperature is too high, the primers will be nonspecific PCR products. On the other hand, when the annealing temperature is too low, the primers will have a hairpin formed at the 3' end.

The optimal melting temperature for the primer pair is based on the GC content of the primers. The GC content should be equal to the product being amplified. However, if the GC content of the primers is higher than that of the product, it is possible that they will form a self-dimer.

The optimal annealing temperature is empirically discovered. It is generally less than the melting temperature of the annealing primers by 5 to 10 degrees. It is recommended to anneal the primers at temperatures between 3degC and 9degC above reaction temperature. This enables the primers to anneal to fully complementary sequences in the target.

Hairpins are commonly formed because the melting temperature of the annealing strand is lower than the melting temperature of the primer. They are usually tolerated at the 3' end, but may be more difficult to break when they are formed at the internal termini of the primer.

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