Primer (molecular biology): Difference between revisions

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{{Short description|Short strand of RNA or DNA that serves as a starting point for DNA synthesis}}

A '''primer''' is a [[nucleic acid]] strand, or a related [[molecule]] that serves as a starting point for [[DNA replication]]. A primer is required because most [[DNA polymerase]]s, [[enzyme]]s that catalyze the replication of [[DNA]], cannot begin synthesizing a new DNA strand from scratch, but can only add to an existing strand of [[nucleotide]]s.

{{Other uses|Primer (disambiguation){{!}}Primer}}

[[File:DNA_replication en.svg|thumb|500px|right|The DNA replication fork. RNA primer labeled at top.]]

A '''primer''' is a short, single-stranded [[nucleic acid]] used by all living organisms in the initiation of [[DNA replication|DNA synthesis]]. A [[Oligonucleotide synthesis|synthetic]] primer is a type of '''[[oligonucleotide|oligo]]''', short for oligonucleotide. [[DNA polymerases]] (responsible for DNA replication) are only capable of adding [[nucleotide]]s to the [[Directionality (molecular biology)|3’-end]] of an existing nucleic acid, requiring a primer be bound to [[DNA|the template]] before DNA polymerase can begin a complementary strand.<ref name="MolecularBiology2016">{{Cite book |editor-last1=Cox |editor-first1=Michael M. |editor-last2=Doudna |editor-first2=Jennifer |editor-last3=O'Donnell |editor-first3=Michael |title=Molecular Biology: Principles and practice |publisher=W. H. Freeman |date=December 21, 2016 |isbn=9781319116378 |language=English}}</ref>

DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replaced with DNA nucleotides. This forms a gap region known as a nick that is filled in using a [[ligase]].<ref>{{Cite journal|last=Henneke|first=Ghislaine|date=2012-09-26|title=In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways|url=https://doi.org/10.1042/BJ20120959|journal=Biochemical Journal|volume=447|issue=2|pages=271–280|doi=10.1042/BJ20120959|pmid=22849643|issn=0264-6021}}</ref> The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides.

In most natural [[DNA replication]], the ultimate primer for DNA synthesis is a short strand of [[RNA]]. This RNA is produced by [[primase]], and is later removed and replaced with DNA by a DNA polymerase.

ManyLiving organisms use solely RNA primers, while laboratory techniques ofin [[biochemistry]] and [[molecular biology]] that involverequire [[in vitro]] DNA polymerases,synthesis (such as [[DNA sequencing]] and [[polymerase chain reaction]] (PCR), requireusually use DNA primers, since they are more temperature stable. ThePrimers primerscan usedbe designed in laboratory for thesespecific techniquesreactions such as polymerase chain reaction (PCR). When designing PCR primers, there are usuallyspecific measures that must be taken into shortconsideration, chemicallylike synthesizedthe DNAmelting moleculestemperature withof athe lengthprimers aboutand twentythe bases.annealing temperature of the reaction itself.

== RNA primers ''in vivo'' ==

{{Further|DNA polymerase|DNA replication}}RNA primers are used by living organisms in the [[Initiation of DNA replication|initiation]] of [[DNA synthesis|synthesizing]] a strand of [[DNA]]. A class of enzymes called [[primase]]s add a complementary RNA primer to the reading template ''[[De novo synthesis|de novo]]'' on both the [[Leading strand|leading]] and [[lagging strand]]s. Starting from the free 3’-OH of the primer, known as the primer terminus, a DNA polymerase can extend a newly synthesized strand. The [[leading strand]] in DNA replication is [[DNA synthesis|synthesized]] in one continuous piece moving with the [[replication fork]], requiring only an initial RNA primer to begin synthesis. In the lagging strand, the template DNA runs in the [[Directionality (molecular biology)|5′→3′ direction]]. Since [[DNA polymerase]] cannot add bases in the 3′→5′ direction complementary to the template strand, DNA is synthesized ‘backward’ in short fragments moving away from the replication fork, known as [[Okazaki fragments]]. Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers. Along the DNA template, [[primase]] intersperses RNA primers that DNA polymerase uses to synthesize DNA from in the 5′→3′ direction.<ref name="MolecularBiology2016" />

Another example of primers being used to enable DNA synthesis is [[Reverse transcriptase|reverse transcription]]. Reverse transcriptase is an enzyme that uses a template strand of RNA to synthesize a complementary strand of DNA. The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis.<ref name="MolecularBiology2016" />

==PCR =Primer designremoval===

After the insertion of [[Okazaki fragments]], the RNA primers are removed (the mechanism of removal differs between [[prokaryote]]s and [[eukaryote]]s) and replaced with new [[deoxyribonucleotides]] that fill the gaps where the RNA primer was present. [[DNA ligase]] then joins the fragmented strands together, completing the synthesis of the lagging strand.<ref name="MolecularBiology2016" />

In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches the previous RNA primer. Then the enzyme simultaneously acts as a [[Phosphodiesterase I|5′→3′ exonuclease]], removing primer [[ribonucleotide]]s in front and adding [[deoxyribonucleotides]] behind. Both the activities of polymerization and excision of the RNA primer occur in the [[Upstream and downstream (DNA)|5′→3′]] direction,  and polymerase I can do these activities simultaneously; this is known as “Nick Translation”.<ref name="MolecularBiology2016" /> Nick translation refers to the synchronized activity of polymerase I in removing the RNA primer and adding [[deoxyribonucleotides]]. Later, a gap between the strands is formed called a nick, which is sealed using a [[DNA ligase]].

In eukaryotes the removal of RNA primers in the [[lagging strand]] is essential for the completion of replication. Thus, as the lagging strand being synthesized by [[DNA polymerase δ]] in [[Upstream and downstream (DNA)|5′→3′]] direction, [[Okazaki fragments]] are formed, which are discontinuous strands of DNA. Then, when the DNA polymerase reaches to the 5’ end of the RNA primer from the previous Okazaki fragment, it displaces the 5′ end of the primer into a single-stranded RNA flap which is removed by nuclease cleavage. Cleavage of the RNA flaps involves three methods of primer removal.<ref name="Uhler2015">{{Cite journal |last1=Uhler |first1=Jay P. |last2=Falkenberg |first2=Maria |date=2015-10-01 |title=Primer removal during mammalian mitochondrial DNA replication |journal=DNA Repair |language=en |volume=34 |pages=28–38 |doi=10.1016/j.dnarep.2015.07.003 |pmid=26303841 |issn=1568-7864|doi-access=free }}</ref> The first possibility of primer removal is by creating a short flap that is directly removed by [[flap structure-specific endonuclease 1]] (FEN-1), which cleaves the 5’ overhanging flap. This method is known as the short flap pathway of RNA primer removal.<ref name="Balakrishnan2013">{{Cite journal |last1=Balakrishnan |first1=Lata |last2=Bambara |first2=Robert A. |date=2013-02-01 |title=Okazaki fragment metabolism |journal=Cold Spring Harbor Perspectives in Biology |volume=5 |issue=2 |pages=a010173 |doi=10.1101/cshperspect.a010173 |issn=1943-0264 |pmc=3552508 |pmid=23378587}}</ref> The second way to cleave a RNA primer is by degrading the RNA strand using a [[RNase]], in eukaryotes it’s known as the RNase H2. This enzyme degrades most of the annealed RNA primer, except the nucleotides close to the 5’ end of the primer. Thus, the remaining nucleotides are displayed into a flap that is cleaved off using FEN-1. The last possible method of removing RNA primer is known as the long flap pathway.<ref name="Balakrishnan2013" /> In this pathway several enzymes are recruited to elongate the RNA primer and then cleave it off. The flaps are elongated by a 5’ to 3’ [[helicase]], known as [[PIF1 5'-to-3' DNA helicase|Pif1]]. After the addition of nucleotides to the flap by Pif1, the long flap is stabilized by the [[replication protein A]] (RPA). The RPA-bound DNA inhibits the activity or recruitment of FEN1, as a result another nuclease must be recruited to cleave the flap.<ref name="Uhler2015" /> This second nuclease is [[Okazaki fragments#Dna2 endonuclease|DNA2 nuclease]], which has a helicase-nuclease activity, that cleaves the long flap of RNA primer, which then leaves behind a couple of nucleotides that are cleaved by FEN1. At the end, when all the RNA primers have been removed, nicks form between the [[Okazaki fragments]] that are filled-in with [[deoxyribonucleotides]] using an enzyme known as [[Ligase|ligase1]], through a process called [[Ligation (molecular biology)|ligation]].

==Uses of synthetic primers==

[[File:Primers RevComp.svg|thumb|Diagrammatic representation of the forward and reverse primers for a standard [[Polymerase chain reaction|PCR]]]]

[[DNA sequencing]] 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.

{{for-multi|the organic chemistry involved|Oligonucleotide synthesis|possible methods involving primers|Nucleic acid methods}}

Synthetic primers are [[Oligonucleotide synthesis|chemically synthesized oligonucleotides]], usually of DNA, which can be customized to [[Annealing (biology)|anneal]] to a specific site on the template DNA. In solution, the primer spontaneously [[Nucleic acid hybridization|hybridizes]] with the template through [[Base pair|Watson-Crick base pairing]] before being extended by DNA polymerase. Both [[Sanger sequencing]] and [[next-generation sequencing]] require primers to initiate the reaction.<ref name="MolecularBiology2016" />

In [[polymerase chain reaction]], 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 nucleotides, and they match exactly the beginning and the end of the DNA fragment to be amplified. They [[Annealing (biology)|anneal]] (adhere) to the DNA template at these starting and ending points, where DNA polymerase binds and begins the synthesis of the new DNA strand.

===PCR primer design===

It is worth noting that primers are not essentially always necessiary for DNA synthesis and can infact be used by viral polymerases, e.g. influenza, for RNA synthesis.

The [[polymerase chain reaction]] (PCR) uses a pair of custom primers to direct DNA elongation toward each other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length and are complementary to the specific upstream and downstream sites of the sequence being amplified.<ref name="MolecularBiology2016" />

Pairs of primers are designed to have similar [[DNA melting|melting temperatures]] since annealing during PCR occurs for both strands simultaneously. The melting temperature is not be either too much higher or lower than the reaction's [[Annealing (biology)|annealing temperature]]. If annealing temperatures are too low, non-specific structures can form, reducing the efficiency of the reaction.<ref>{{cite journal |last1=Bakhtiarizadeh |first1=Mohammad Reza |last2=Najaf-Panah |first2=Mohammad Javad |last3=Mousapour |first3=Hojatollah |last4=Salami |first4=Seyed Alireza |title=Versatility of different melting temperature (Tm) calculator software for robust PCR and real-time PCR oligonucleotide design: A practical guide |journal=Gene Reports |date=March 2016 |volume=2 |pages=1–3 |doi=10.1016/j.genrep.2015.11.001}}</ref>

==PCR Primer design==

The choice of the length of the primers and their [[DNA melting|melting temperature (''T''_m)]] depends on a number of considerations. The [[DNA melting|melting temperature]] of a primer is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature required increases with the length of the primer. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the temperature required to melt it. Melting temperatures that are too high, i.e., above 80 °C, can also cause problems since the DNA polymerases used for PCR are less active at such temperatures. The optimum length of a primer is generally from 20 to 30 nucleotides with a melting temperature between about 55 °C and 65 °C.

Additionally, primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of hybridization to a similar sequence nearby. A commonly used method for selecting a primer site is [[BLAST (biotechnology)|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 [[National Center for Biotechnology Information|NCBI]] tool Primer-BLAST integrates primer design and BLAST search into one application,<ref>{{cite web| url = https://www.ncbi.nlm.nih.gov/tools/primer-blast/| title = Primer-BLAST |website=NCBI}}</ref> as do commercial software products such as ePrime and [[Beacon Designer]]. [[In silico PCR|''In silico'' PCR]] may be performed to evaluate the specificity of designed primers.<ref>{{cite journal |last1=Kalendar |first1=Ruslan |last2=Shevtsov |first2=Alexandr |last3=Otarbay |first3=Zhenis |last4=Ismailova |first4=Aisulu |title=In silico PCR analysis: a comprehensive bioinformatics tool for enhancing nucleic acid amplification assays |journal=Frontiers in Bioinformatics |date=7 October 2024 |volume=4 |doi=10.3389/fbinf.2024.1464197 |pmid=39435190 |doi-access=free|pmc=11491563 }}</ref>

Pairs of primers should have the similar melting temperatures as annealing in a PCR reaction occurs for both simultaneously. A primer with a ''T''_m significantly higher than the reaction's annealing temperature my mishybridize and extend at an incorrect ___location along the DNA sequence, while ''T''_m significantly lower than the annealing temperature may fail to anneal and extend at all.

PrimerSelecting sequencesa needspecific toregion beof chosen to uniquely selectDNA for aprimer regionbinding ofrequires DNA,some avoidingadditional theconsiderations. possibilityRegions ofhigh mishybridizationin tomononucleotide a similar sequence nearby.and Mononucleotidedinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers shouldthat notare easilycomplementary annealto witheach other primerscan inlead to the mixtureformation (eitherof otherprimer-dimers, copieslowering ofthe sameefficiency orof the reversedesired directionreaction.<ref primer);name=Principles2008>{{cite thisbook phenomenon|title=Principles canand leadTechnical toAspects theof productionPCR ofAmplification [[primer|date=2008 dimer]]|publisher=Springer productsNetherlands contaminating|isbn=978-1-4020-6241-4 the|pages=63–90 mixture|chapter-url=https://doi.org/10.1007/978-1-4020-6241-4_5 |language=en |chapter=PCR Primers should|doi=10.1007/978-1-4020-6241-4_5 also}}</ref> notPrimers annealthat stronglyare able to anneal to themselves, ascan form internal hairpins and loops couldthat hinder the annealinghybridization with the template DNA.<ref name=Principles2008 />

When designing primers, additional nucleotide bases can be added to the back ends of each primer, resulting in a customized cap sequence on each end of the amplified region. One application for this practice is for use in [[TA cloning]], a special subcloning technique similar to PCR, where efficiency can be increased by adding AG tails to the 5′ and the 3′ ends.<ref>{{cite journal |last1=Peng |first1=Ri-He |last2=Xiong |first2=Ai-Sheng |last3=Liu |first3=Jin-ge |last4=Xu |first4=Fang |last5=Bin |first5=Cai |last6=Zhu |first6=Hong |last7=Yao |first7=Quan-Hong |title=Adenosine added on the primer 5′ end improved TA cloning efficiency of polymerase chain reaction products |journal=Analytical Biochemistry |date=April 2007 |volume=363 |issue=1 |pages=163–165 |doi=10.1016/j.ab.2007.01.014 |pmid=17303063 }}</ref>

===Degenerate primers===

{{main|Degenerate bases}}

SometimesSome situations may call for the use of ''degenerate primers.'' are used. These are actually mixtures of primers that are similar, but not identical, primers. TheyThese may be convenient ifwhen amplifying the same [[gene]] is to be amplified from different [[organism]]s, as the genes themselvessequences are probably similar but not identical. TheThis othertechnique useis foruseful degeneratebecause primersthe is[[genetic whencode]] primer designitself is based on [[proteinDegenerate sequencecode|degenerate]]., Asmeaning several different [[codon]]s can code for onethe same [[amino acid]],. itThis isallows oftendifferent difficultorganisms to deducehave whicha codonsignificantly isdifferent usedgenetic insequence that code for a particularhighly casesimilar protein. For this reason, degenerate primers are also used when primer design is based on [[protein sequence]], as the specific sequence of codons are not known. 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]]., Useusing ofthe IUPAC symbols for [[degenerate primersbases]]. canDegenerate greatlyprimers reducemay thenot specificityperfectly ofhybridize thewith PCRa amplification.target Thesequence, problemwhich can begreatly partlyreduce solvedthe byspecificity usingof [[touchdownthe PCR]] amplification.

''Degenerate primers'' are widely used and extremely useful in the field of [[microbial ecology]]. TheThey allow for the amplification of genes from thus far uncultivated microorganisms[[microorganism]]s 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 of the codon sequence.

==Oligonucleotide synthesis==

The actual construction of such primers starts with 3'-hydroxyl nucleosides ([[phosphoramidite]]) attached to a [[controlled-pore glass]] (CPG). The 5'-hydroxyl of the nucleosides is covered [[dimethoxytrityl]] (DMT), which prevents the building of a nucleotide chain. To add a nucleotide, DMT is chemically removed, and the nucleotide is added. The 5'-hydroxyl of the new nucleotide is blocked by DMT, preventing the addition of more than one nucleotide to each chain. After that, the cycle is repeated for each nucleotide in the primer. This is a simplified description; the actual process is quite complicated. For that reason, most laboratories do not make primers themselves, but order them by specialized companies.

==References==

{{Reflist}}

An earlier version of the above article was posted on ''[http://nupedia.8media.org Nupedia]''.

[[Category:Molecular biology]]

==External links==

* [http://frodo.wi.mit.edu/primer3/ Primer3]

* [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer3Plus] -- Easy and powerful primer design, based on primer3

* [https://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST]

* [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Primer 3] -- Primer3 -- Online primer design software

<!--Please do not add more links to this section without first discussing them on the talk page. Wikipedia is not an indiscriminate repository of links.-->

* [http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm FastPCR] PCR primer/probe design software

* [http://www.changbioscience.com/primo/ Primo] -- Online primer design

* [http://www.genome.ou.edu/informatics/primou.html] -- PrimOU primer design program

* [http://www.biophp.org/minitools/melting_temperature/demo.php TM calculation] - by bioPHP.org.

* {{MeshName|DNA+primers}}

* {{MeshName|RNA+primers}}

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