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Primer dimer is formed and amplified in a three-step process. A primer dimer is formed and amplified in three steps. Primer dimers may be visible after gel electrophoresis of the PCR product. 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. One approach to prevent PDs consists of physical-chemical optimization of the PCR system, i. 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.
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C, they have some polymerizing activity also at lower temperatures, which can cause DNA synthesis from primers after annealing to each other. Wax: in this method the enzyme is spatially separated from the reaction mixture by wax that melts when the reaction reaches high temperature. Cold-sensitive Taq polymerase: is a modified DNA polymerase with almost no activity at low temperature. Chemical modification: in this method a small molecule is covalently bound to the side chain of an amino acid in the active site of the DNA polymerase.
Another approach to prevent or reduce PD formation is by modifying the primers so that annealing with themselves or each other does not cause extension. 3′ end of the primer is added to the 5′ end of the primer. Because of the close proximity of the 5′ tail it anneals to the 3′ end of the primer. Chimeric primers: some DNA bases in the primer are replaced with RNA bases, creating a chimeric sequence.
The melting temperature of a chimeric sequence with another chimeric sequence is lower than that of chimeric sequence with DNA. This difference enables setting the annealing temperature such that the primer will anneal to its target sequence, but not to other chimeric primers. RNase HII to remove a blocking group from the PCR primers at high temperature. This RNase HII enzyme displays almost no activity at low temperature, making the removal of the block only occur at high temperature. While the methods above are designed to reduce PD formation, another approach aims to minimize signal generated from PDs in quantitative PCR. This approach is useful as long as there are few PDs formed and their inhibitory effect on product accumulation is minor.