...

from amplified primer-dimers and primer-oligomers. The concentration of primers is much higher than that of target DNA in PCR. Therefore, if the primers exhibit self-complementarity they may hybridize to each other and form homo-dimers or hetero-dimers. Avoiding primers with 3′ overlaps is extremely important in multiplex reactions. A self-dimer is formed due to intermolecular interactions of the same primer. A cross dimer is formed due to intermolecular interaction between sense and antisense primers. In order to detect cross dimers, the sense primer in 5′ - 3′ direction is compared with the antisense primer in 3′ - 5′ direction for homology. A Primer dimer (PD) is a potential by-product in PCR, a common biotechnological method. As its name implies, a PD consists of primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification. Detection Primer dimers may be visible after gel electrophoresis of the PCR product. PDs in ethidium bromide-stained gels are typically seen as a 30-50 base-pair (bp) band or smear of moderate to high intensity and distinguishable from the band of the target sequence, which is typically longer than 50 bp. In quantitative PCR, PDs may be detected by melting curve analysis with intercalating dyes, such as SYBR Green I, a nonspecific dye for detection of double-stranded DNA. Because they usually consist of short sequences, the PDs denaturate at lower temperature than the target sequence and hence can be distinguished by their melting-curve characteristics. Preventing primer-dimer formation One approach to prevent PDs consists of physical-chemical optimization of the PCR system, i.e., changing the concentrations of primers, magnesium chloride, nucleotides, ionic strength and temperature of the reaction. This method is somewhat limited by the physical-chemical characteristics that also determine the efficiency of amplification of the target sequence in the PCR. Therefore, reducing PDs formation may also result in reduced PCR efficiency. To overcome this limitation, other methods aim to reduce the formation of PDs only, including primer design, and use of different PCR enzyme systems or reagents. Primers-design software Primer-design software uses algorithms that check for the potential of DNA secondary structure formation and annealing of primers to itself or within primer pairs. Physical parameters that are taken into account by the software are potential self-complementarity and GC content of the primers; similar melting temperatures of the primers; and absence of secondary structures, such as stem-loops, in the DNA target sequence.