A primer dimer (PD) is a potential by-product in the
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR), a common biotechnological method. As its name implies, a PD consists of two
primer
Primer may refer to:
Arts, entertainment, and media Films
* ''Primer'' (film), a 2004 feature film written and directed by Shane Carruth
* ''Primer'' (video), a documentary about the funk band Living Colour
Literature
* Primer (textbook), a t ...
molecules that have attached (
hybridized) to each other because of strings of
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
bases in the primers. As a result, the
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the
DNA sequence targeted for PCR amplification. In
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
, PDs may interfere with accurate quantification.
Mechanism of formation
![Primer dimers formation En](https://upload.wikimedia.org/wikipedia/commons/1/19/Primer_dimers_formation_En.PNG)
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
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
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
In molecular biology and genetics, GC-content (or guanine-cytosine content) is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). This measure indicates the proportion of G and C bases out o ...
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.
Detection
Primer dimers may be visible after
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
of the PCR product. PDs in
ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
-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
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
, PDs may be detected by
melting curve analysis
Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. ...
with intercalating dyes, such as
SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
, a nonspecific dye for detection of double-stranded DNA. Because they usually consist of short sequences, the PDs denature 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
Magnesium chloride is the family of inorganic compounds with the formula , where x can range from 0 to 12. These salts are colorless or white solids that are highly soluble in water. These compounds and their solutions, both of which occur in natu ...
,
nucleotides
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules w ...
,
ionic strength
The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic compounds, when dissolved in water, dissociate into ions. The total electrolyte concentration in solution will affect important properties such as ...
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.
Primer-design software
Primer-design software uses algorithms that check for the potential of DNA
secondary structure
Protein secondary structure is the three dimensional conformational isomerism, form of ''local segments'' of proteins. The two most common Protein structure#Secondary structure, secondary structural elements are alpha helix, alpha helices and beta ...
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-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when ...
s, in the DNA target sequence.
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. 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.
''Slow release of magnesium'': DNA polymerase requires magnesium ions for activity, 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
''Non-covalent binding of inhibitor'': in this method a
peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A ...
,
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
or
aptamer
Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities ( KD in the pM to μM range), with little or no off-target binding ...
are
non-covalently bound to the enzyme at low temperature and inhibit its activity. After an incubation of 1–5 minutes at 95 °C, the inhibitor is released and the reaction starts.
''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
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called the "main chain" or backbone. The side chain is a hydrocarbon branching element of a molecule that is attached to a l ...
of an
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
in the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
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.
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): 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
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when ...
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.
''Blocked-cleavable primers'': a method known as
RNase H-dependent PCR (rhPCR),
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, 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, 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
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
. This approach is useful as long as there are few PDs formed and their inhibitory effect on product accumulation is minor.
''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.
Four steps PCR
/ref>
''Sequence-specific probes'': TaqMan TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann ...
and molecular beacon
Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluo ...
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
External links
{{DEFAULTSORT:Primer dimer
Molecular biology
Biotechnology
Polymerase chain reaction
Laboratory techniques