Primer dimer
A primer dimer (PD) is a potential by-product in the
Mechanism of formation
A primer dimer is formed and amplified in three steps. In the first step, two primers anneal at their respective 3' ends (step I in the figure). If this construct is stable enough, the DNA polymerase will bind and extend the primers according to the complementary sequence (step II in the figure). An important factor contributing to the stability of the construct in step I is a high GC-content at the 3' ends and length of the overlap. The third step occurs in the next cycle, when a single strand of the product of step II is used as a template to which fresh primers anneal leading to synthesis of more PD product.[1]
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
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,
Primer-design software
Primer-design software uses algorithms that check for the potential of DNA
Hot-start PCR
Because primers are designed to have low complementarity to each other, they may anneal (step I in the figure) only at low temperature, e.g. room temperature, such as during the preparation of the reaction mixture. Although DNA polymerases used in PCR are most active around 70 °C, they have some polymerizing activity also at lower temperatures, which can cause DNA synthesis from primers after annealing to each other.[3] Several methods have been developed to prevent PDs formation until the reaction reaches working temperature (60-70 °C), and these include initial inhibition of the DNA polymerase, or physical separation of reaction components reaction until the reaction mixture reaches the higher temperatures. These methods are referred to as hot-start PCR.
Wax: in this method the enzyme is spatially separated from the reaction mixture by wax that melts when the reaction reaches high temperature.[4]
Slow release of magnesium: DNA polymerase requires magnesium ions for activity,[5] so the magnesium is chemically separated from the reaction by binding to a chemical compound, and is released into the solution only at high temperature [6]
Non-covalent binding of inhibitor: in this method a
Cold-sensitive Taq polymerase: is a modified DNA polymerase with almost no activity at low temperature.[9]
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. The small molecule is released from the enzyme by incubation of the reaction mixture for 10–15 minutes at 95 °C. Once the small molecule is released, the enzyme is activated.[10]
Structural modifications of primers
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.
HANDS (Homo-Tag Assisted Non-Dimer System[11]): a nucleotide tail, complementary to the 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. The result is a stem-loop primer that excludes annealing involving shorter overlaps, but permits annealing of the primer to its fully complementary sequence in the target.
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.[12]
Blocked-cleavable primers: a method known as RNase H-dependent PCR (rhPCR),[13] utilizes a thermostable 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. The enzyme also possess inherent primer:template mismatch discrimination, resulting in additional selection against primer-dimers.
Self-Avoiding molecular recognition systems :also known as SAMRS,[14] eliminating primer dimers by introducing nucleotide analogues T*, A*, G* and C* into the primer. The SAMRS DNA could bind to natural DNA, but not to other members of the same SAMRS species. For example, T* could bind to A but not A*, and A* could bind to T but not T*. Thus, through careful design,[15] primers build from SAMRS could avoid primer-primer interactions and allowing sensitive SNP detection as well as multiplex PCR.
Preventing signal acquisition from primer dimers
While the methods above are designed to reduce PD formation, another approach aims to minimize signal generated from PDs in
Four steps PCR: used when working with nonspecific dyes, such as SYBR Green I. It is based on the different length, and hence, different melting temperature of the PDs and the target sequence. In this method the signal is acquired below the melting temperature of the target sequence, but above the melting temperature of the PDs.[16]
Sequence-specific probes: TaqMan and molecular beacon probes generate signal only in the presence of their target (complementary) sequence, and this enhanced specificity precludes signal acquisition (but not possible inhibitory effects on product accumulation) from PDs.
References
- ^ Alberts; et al. (2017). Molecular Biology of the Cell (6th ed.). Garland Science. pp. 708–711.
- ^ The primer design page of Leiden University Medical Center
- ^ Patel, Ewing (2008). Polymerase Chain Reaction: Techniques and Applications. Scientific Press. pp. 595–599.
- PMID 1579465.
- PMID 15238001.
- ^ US Patent application number 2007/0254327
- ^ US Patent number 5338671
- ^ US Patent number 6183967
- ^ US Patent number 6214557
- ^ US Patent number 5677152
- PMID 9241236.
- Patent Lens.
- PMID 21831278.
- PMID 20586087.
- PMID 32395633.
- ^ Four steps PCR
External links
"Online software for primer dimer prediction". OligoAnalyzer 3.1. Integrated DNA Technologies.
"Primer design. What is the primer-dimer?". YouTube video. Archived from the original on 2021-12-20.