Primers primer-dimer

Primer design is one of the most important aspects of a robust PCR assay. In general, primers should be designed such that they are not able to form secondary structures such as stemloop or hairpin configurations. A primer must not be complementary at the 3 end, as this will cause primer dimers to form. All primers should have similar melting temperatures and should not contain stretches of individual nucleotides. There are software programs available to assist in primer design, but it is crucial that primers are tested in the assay, especially in a multiplex system. 

Attention should be given to the selection of primers. Typically, primers used are between 15 and 30 bases in length, with guanine-cytosine composition between 40 and 60%. The primer should not have within its sequence any unusual composition such as stretches of polypurines or polypyrimidines. The primer pair should not be complementary at the 3 ends, since otherwise the DNA synthesizing enzyme can extend one primer over the other primer, creating a double-stranded product whose length approximates the sum of the two primers. This artifact is called primer dimer, which could very well become the predominant and undesirable PCR product when primer pairs complementary at the 3 ends are used (B4, N3). 

In this approach, bisulfite-treated DNA is used as the template, and 50-100nM nnmethylated or methylated DNA-specific primers are nsed for PCR amplification in separate reactions. For quantification, a AAC.J, method is nsed and normalized with the C.J, (cycle threshold) for the 3-actin gene (43). Alternatively, a fragment of the target gene promoter will be amplified using primers designed from a CpG-free area as an internal control (see Note 3). To eliminate any primer dimer that will compromise the accuracy of the results, an additional step in the PCR cycles above... 

For QM-MSP, first the promoter regions of several genes will be amplified using primes that correspond to the CG-free regions (45). Then, 1 pL of the diluted PCR product (30-50 times) is used as the template in real-time QM-MSP to quantify the methylated and unmethylated template in separate reactions using methylation- or nonmethylation-specific primers. The benefits of this method are that (1) the instability of bisulfite-modified DNA is remedied using an initial round of PCR (2) nonspecific products that compromise the accuracy of SYBR green-based real-time PCR are eliminated in the second-round PCR (3) the likelihood of the development of primer dimers is minimized as the first-round PCR product is diluted 50 times and a minimal amount of MSP primers is used in the second-round PCR (4) the amount of the precious DNA used in this approach is substantially less than in the other methods. 

Fig. 2. Amplification of ORFs by 2-step PCR. The products of the second PCR were elec-trophoresised using a 1% agarose gel. There are some ORFs having the same 5 - and 3 - sequences, although the internal sequences are distinct (marked by ). This property is typical in wttgenes (6). These ORFs were simultaneously amplified by the same set of primers and distinguished by sequencing after cloning. Primer dimers are sometimes observed when a desired ORF is not amplified ( ). M k/Flindll marker.

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. 

Since DNA fragments from the PCR typically are contained in a high salt matrix, their mobility will vary depending on sample salt concentration. Thus, proper identification of these DNA fragments requires the use of an internal standard to normalize analyte velocity. This practice corrects for variance in fragment mobility due to sample matrix differences (i.e., salt content). These internal standards are included for size determination (in bp) as well as a reference for migration time. Candidates for such internal standards include the primer or primer-dimer peaks, since both components are already present in the PCR mixture alternatively, one or more coinjected standard DNA peak s may be chosen. If any of these fragments are to serve as the internal standard, they must be separated from one another and any PCR product, a precondition that is not easily met when the size of the PCR product is below 60 bp. 

Figure 7.11 CE-LIF separation and quantitation of fluorescein-labeled lambda PCR product. A 500 bp product was generated with titrated amounts of lambda bacteriophage template and primers, one of which was fluorescein-labeled. The fragment was analyzed by CE-LIF, and only the fiuor from the PCR product was detected. With increasing amounts of DNA template, peak height and area of the product also increased up to a point, whereupon the PCR plateaued. Note the increase in primer and primer-dimer peaks as template availability decreases. (Reproduced with permission from KJ Ulfelder, Applications Information Bulletin A-1774, 1994. Copyright Beckman Instruments, Inc.)...

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. 

One advantage of fluorescently labeled primers over dsDNA dyes is that multiplexing is possible. However, with both dsDNA dyes and labeled primers, reaction specificity depends on the specificity of the primers. Any double-stranded product that is formed will be detected, including primer-dimers. Therefore, hot start techniques, temperature discrimination by collecting real-time data at a high temperature, and melting curve analysis to confirm the desired product are useful. 

Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res 1997 25 3235-41. 

Non-hot start Taq is also snitable however, primer dimers are less of a problem with hot start Taq and this is recommended. 

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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