Primer dimer
Encyclopedia
A Primer dimer 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 real-time PCR, PDs may interfere with accurate quantification.
will bind and extend the primers according to the complementary sequence (step II in the figure). Main factors 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.
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. However, this method is unfavored due to the high risk of contamination of the lab with PCR product.
In real-Time 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.
, 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.
s, in the DNA target sequence.
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
, antibody
or aptamer
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
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.
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
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.
While the methods above are designed to reduce PD formation, another approach aims to minimize signal generated from PDs in Real-Time PCR. 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.
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.
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
molecules that have attached (hybridized
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
) to each other because of strings of complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
bases
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
in the primers. As a result, the DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
sequence targeted for PCR amplification. In real-time PCR, PDs may interfere with accurate quantification.
Mechanism of formation
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 polymeraseTaq polymerase
thumb|228px|right|Structure of Taq DNA polymerase bound to a DNA octamerTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965...
will bind and extend the primers according to the complementary sequence (step II in the figure). Main factors contributing to the stability of the construct in step I is a high GC-content
GC-content
In molecular biology and genetics, GC-content is the percentage of nitrogenous bases on a DNA molecule that are either guanine or cytosine . This may refer to a specific fragment of DNA or RNA, or that of the whole genome...
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 electrophoresisGel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
of the PCR product. PDs in ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
-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. However, this method is unfavored due to the high risk of contamination of the lab with PCR product.
In real-Time PCR, PDs may be detected by melting curve analysis
Melting curve analysis
Melting curve analysis is an assessment of the dissociation-characteristics of double-stranded DNA during heating. The information gathered can be used to infer the presence and identity of single-nucleotide polymorphisms.-Implementation:...
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 chlorideMagnesium chloride
Magnesium chloride is the name for the chemical compounds with the formulas MgCl2 and its various hydrates MgCl2x. These salts are typical ionic halides, being highly soluble in water. The hydrated magnesium chloride can be extracted from brine or sea water...
, nucleotides, ionic strength
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 the dissociation or the solubility of different salts...
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-loopStem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in 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 read in opposite directions,...
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
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
, antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
or aptamer
Aptamer
Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as...
are non-covalently
Noncovalent bonding
A noncovalent bond is a type of chemical bond, typically between macromolecules, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. The noncovalent bond is the dominant type of bond between supermolecules in...
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
Covalent bond
A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
to the side chain
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 "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...
of an amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
in the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
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
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in 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 read in opposite directions,...
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.
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 Real-Time PCR. 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.
Sequence specific probes: Taqman
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
and Molecular beacon
Molecular beacon
Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence...
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.