Primer-dimers formation

Primer dimer formation can be a potential problem in PCR amplifications. Although we generally avoided the use of self-complementary sequences when designing primers, we found that dimer formation was not a problem at an annealing temperature of 35° even when 6 bases of a 10-bp primer could potentially hybridize. 

Hot-start PCR can also reduce the amount of primer-dimer formation by increasing the stringency of primer annealing. At lower temperatures, the primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer-dimer, which can often be observed as a diffuse band of approximately 50 to 100 bp on an ethidium bromide-stained gel. The formation of nonspecific amplification products and primer-dimer can compete for reagent availability with the amplification of the product desired. Therefore, hot-start PCR can improve the yield of the specific PCR products. 

Samples that have very low target mRNA concentrations often display increased background, particularly if amplification above 35 cycles is required. The initial formation of these products can occur at low initial (ambient) temperatures, and are reduced or eliminated by the hot start approach, in which wax beads are added, melted to form a solid layer above the cDNA, and then the PCR master mix added on top. The two solutions are mixed and the PCR reaction begins after the thermocycler heats to 94°C. Background is sometimes due to the formation of primer dimers, a double-stranded PCR product consisting of the two primers and their complementary sequences. Sometimes these primer dimers contain extra sequences between the primers (14). When primer dimers form, they can be a major problem as they are very efficiently amplified and compete with amplification of the target cDNA. This is not a major problem with the primer sets described in Table 1, but can be a problem with other primer sets. With persistent problems, the hot start approach could be modified to add the cDNA, the primers, and the Taq polymerase separately. 

No possible dimer formation with other primers used in multiplex PCR. 

The advantage of real-time PCR over traditional PCR is the ability to quantify DNA content in the sample. It provides a higher degree of specificity given by a fluorescent probe which anneal in the sequence flanked by the two primers. It also reduces interferences due to the formation of primer dimers and nonspecific products (Brzezinski, 2006). RT-PCR is less prone to contamination and is suitable for automation. Numerous assays based on RT-PCR have been developed for the detection of traces of food allergens in raw materials and processed foods (Table 9.1). 

The set of primers has to be designed specifically for the sample DNA. The primer length is usually 10-30 base pairs (bp) and their complementary sequence must be unique in the template. Additionally, there should be no intra or inter primer complementarity in order to avoid the formation of primer-dimers. Ideally, the number of each base in the primer is relatively equal. Unusual sequences such as long stretches of polypurines or polypyrimidines and repetitive sequences must be avoided. The melting temperature for both primers should be similar and lie between 55 and 80 °C. 

Duplex Formation If the internal primer can dimerize with itself, as for the mis-priming, unacceptable background may result, or reduced signal intensity owing to snb-optimum primer annealing. 

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