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Primer (molecular biology)
Encyclopedia
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 enzyme
s that catalyze this process, DNA polymerase
s, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end
of the primer, and copies the opposite strand
.
In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA
(which can be made de novo
).
Many of the laboratory techniques of biochemistry
and molecular biology
that involve DNA polymerase, such as DNA sequencing
and the polymerase chain reaction
(PCR), require DNA primers. These primers are usually short, chemically synthesized
oligonucleotides, with a length of about twenty bases. They are hybridized to a target DNA, which is then copied by the polymerase.
The lagging strand is that strand of the DNA double helix that is orientated in a 5' to 3' manner. Therefore, its complement must be synthesized in a 3'→5' manner. Because DNA polymerase III cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragment
s. Along the lagging strand's template, primase
builds RNA primers in short bursts. DNA polymerases are then able to use the free 3'-OH
groups on the RNA primers to synthesize DNA in the 5'→3' direction.
The RNA fragments are then removed by DNA polymerase I
for prokaryotes or DNA polymerase δ for eukaryotes (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase
then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.
direction, and when it encounters the RNA primer from the previous Okazaki fragment, displacing the 5′ end of the primer into a single-stranded RNA flap, which is removed by nuclease cleavage. Cleavage of the RNA flaps involves either endonuclease 1 (FEN1) cleavage of short flaps, or coating of long flaps by the single-stranded DNA binding protein replication protein A
(RPA) and sequential cleavage by Dna2 nuclease and FEN1.
This mechanism is a potential explanation to how HIV
virus can transform its genome into double stranded DNA from the RNA-DNA formed after reverse transcription of its RNA. However, the HIV-encoded reverse transcriptase
has own ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense
cDNA strand into an antisense DNA to form a double-stranded DNA intermediate.
is used to determine the nucleotides in a DNA strand; the chain termination method (dideoxy sequencing or Sanger method) uses a primer as a start marker for the chain reaction.
In PCR
, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 (usually 18–24) nucleotides, and they need to match the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other – the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment.
It is worth noting that primers are not essentially always necessary for DNA synthesis and can in fact be used by viral polymerases, e.g. influenza, for RNA synthesis.
Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is BLAST
search whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI
tool Primer-BLAST integrates primer design tool and BLAST search into one application, so does commercial software product such as Beacon Designer
. Mononucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.
When designing a primer for use in TA cloning
, efficiency can be increased by adding AG tails to the 5' and the 3' end.
The reverse primer has to be the reverse complement of the given cDNA sequence. The reverse complement can be easily determined, e.g. with on-line calculators.
is to be amplified from different organism
s, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid
, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid
isoleucine
might be "ATH", where A stands for adenine
, T for thymine
, and H for adenine
, thymine
, or cytosine
, according to the genetic code
for each codon, using the IUPAC symbols for degenerate bases. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.
Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank
. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
that serves as a starting point for DNA synthesis
DNA synthesis
DNA synthesis commonly refers to:*DNA replication - DNA biosynthesis *Polymerase chain reaction - enzymatic DNA synthesis *Oligonucleotide synthesis - chemical synthesis of nucleic acids...
. They are required for DNA replication because the enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s that catalyze this process, 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....
s, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end
Directionality (molecular biology)
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5′-end and a 3′-end...
of the primer, and copies the opposite strand
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...
.
In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
(which can be made de novo
De novo synthesis
De novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as...
).
Many of the laboratory techniques of biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
that involve DNA polymerase, such as DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
and the polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR), require DNA primers. These primers are usually short, chemically synthesized
Oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
oligonucleotides, with a length of about twenty bases. They are hybridized to a target DNA, which is then copied by the polymerase.
Mechanism in vivo
The lagging strand is that strand of the DNA double helix that is orientated in a 5' to 3' manner. Therefore, its complement must be synthesized in a 3'→5' manner. Because DNA polymerase III cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragment
Okazaki fragment
Okazaki fragments are short molecules of single-stranded DNA that are formed on the lagging strand during DNA replication. They are between 1,000 to 2,000 nucleotides long in Escherichia coli and are between 100 to 200 nucleotides long in eukaryotes....
s. Along the lagging strand's template, primase
Primase
DNA primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template...
builds RNA primers in short bursts. DNA polymerases are then able to use the free 3'-OH
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
groups on the RNA primers to synthesize DNA in the 5'→3' direction.
The RNA fragments are then removed by DNA polymerase I
DNA polymerase I
DNA Polymerase I is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known...
for prokaryotes or DNA polymerase δ for eukaryotes (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.
Primer removal
In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in 5' to 3'Upstream and downstream (DNA)
In molecular biology and genetics, upstream and downstream both refer to a relative position in DNA or RNA. Each strand of DNA or RNA has a 5' end and a 3' end, so named for the carbons on the deoxyribose ring. Relative to the position on the strand, downstream is the region towards the 3' end of...
direction, and when it encounters the RNA primer from the previous Okazaki fragment, displacing the 5′ end of the primer into a single-stranded RNA flap, which is removed by nuclease cleavage. Cleavage of the RNA flaps involves either endonuclease 1 (FEN1) cleavage of short flaps, or coating of long flaps by the single-stranded DNA binding protein replication protein A
Replication protein A
Replication protein A is a protein that binds single-stranded DNA in eukaryotic cells. During DNA replication, RPA prevents single-stranded DNA from winding back on itself or from forming secondary structures. This keeps DNA unwound for the polymerase to replicate it...
(RPA) and sequential cleavage by Dna2 nuclease and FEN1.
This mechanism is a potential explanation to how HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
virus can transform its genome into double stranded DNA from the RNA-DNA formed after reverse transcription of its RNA. However, the HIV-encoded reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
has own ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense
Sense (molecular biology)
In molecular biology and genetics, sense is a concept used to compare the polarity of nucleic acid molecules, such as DNA or RNA, to other nucleic acid molecules...
cDNA strand into an antisense DNA to form a double-stranded DNA intermediate.
Uses of synthetic primers
DNA sequencingDNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
is used to determine the nucleotides in a DNA strand; the chain termination method (dideoxy sequencing or Sanger method) uses a primer as a start marker for the chain reaction.
In PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 (usually 18–24) nucleotides, and they need to match the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other – the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment.
It is worth noting that primers are not essentially always necessary for DNA synthesis and can in fact be used by viral polymerases, e.g. influenza, for RNA synthesis.
PCR primer design
Pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. A primer with a Tm significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while Tm significantly lower than the annealing temperature may fail to anneal and extend at all.Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. A commonly used method is BLAST
BLAST
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences...
search whereby all the possible regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched. The free NCBI
National Center for Biotechnology Information
The National Center for Biotechnology Information is part of the United States National Library of Medicine , a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper...
tool Primer-BLAST integrates primer design tool and BLAST search into one application, so does commercial software product such as Beacon Designer
Beacon designer
Beacon Designer designs highly specific an efficient primers and probes for real time PCR assays. It is compatible to work on Windows as well as on Mac. The software currently supports the following real time PCR chemistries for efficient primer and probe design.1. SYBR Green 2. TaqMan 3....
. Mononucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.
When designing a primer for use in TA cloning
TA cloning
TA cloning is a subcloning technique that doesn't use restriction enzymes and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine and thymine on different DNA fragments to hybridize and, in the presence of ligase, become ligated together...
, efficiency can be increased by adding AG tails to the 5' and the 3' end.
The reverse primer has to be the reverse complement of the given cDNA sequence. The reverse complement can be easily determined, e.g. with on-line calculators.
Degenerate primers
Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical primers. They may be convenient if the same geneGene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
is to be amplified from different organism
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
s, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one 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...
, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the 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...
isoleucine
Isoleucine
Isoleucine is an α-amino acid with the chemical formula HO2CCHCHCH2CH3. It is an essential amino acid, which means that humans cannot synthesize it, so it must be ingested. Its codons are AUU, AUC and AUA....
might be "ATH", where A stands for adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
, T for thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
, and H for adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
, thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
, or cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
, according to the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
for each codon, using the IUPAC symbols for degenerate bases. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.
Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank
GenBank
The GenBank sequence database is an open access, annotated collection of all publicly available nucleotide sequences and their protein translations. This database is produced and maintained by the National Center for Biotechnology Information as part of the International Nucleotide Sequence...
. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.