Editing Primer (molecular biology)

{{Short description|Short strand of RNA or DNA that serves as a starting point for DNA synthesis}}
{{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 may also be referred to as an '''oligo''', short for oligonucleotide. [[DNA polymerase]] (responsible for DNA replication) enzymes 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=":0">{{Cite book|title=Molecular Biology: Principles and Practice|last=Cox|first=Michael M.|publisher=W. H. Freeman and Company|year=2015|isbn=9781464126147|location=New York|pages=221–238, 369–376, 592–593}}</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 replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called 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. Living organisms use solely RNA primers, while laboratory techniques in [[biochemistry]] and [[molecular biology]] that require [[in vitro]] DNA synthesis (such as [[DNA sequencing]] and [[polymerase chain reaction]]) usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.

== 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=":0" />

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=":0" />

===Primer removal===
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=":0" />

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>{{Cite book |last=Doudna; Cox; O'Donnell |first=Jennifer; Michael M.; Michael |title=Molecular Biology: Principles and practice |publisher=W. H. Freeman |date=December 21, 2016 |isbn=9781319116378 |language=English}}</ref> 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=":1">{{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=":2">{{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=":2" /> 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=":1" /> 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]]]]
{{for-multi|the organic chemistry involved|Oligonucleotide synthesis|possible methods involving primers|Nucleic acid methods}}

Synthetic primers, sometimes known as oligos, 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. The ability to create and customize synthetic primers has proven an invaluable tool necessary to a variety of molecular biological approaches involving the analysis of DNA. Both the [[Sanger sequencing|Sanger chain termination method]] and the “[[DNA sequencing|Next-Gen]]” method of DNA sequencing require primers to initiate the reaction.<ref name=":0" />

===PCR primer design===
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 must code for only the specific upstream and downstream sites of the sequence being amplified. A primer that can bind to multiple regions along the DNA will amplify them all, eliminating the purpose of PCR.<ref name=":0" />

A few criteria must be brought into consideration when designing a pair of PCR primers. Pairs of primers should have similar melting temperatures since annealing during PCR occurs for both strands simultaneously, and this shared melting temperature must not be either too much higher or lower than the reaction's [[Annealing (biology)|annealing temperature]]. A primer with a ''T''<sub>m</sub> (melting temperature) too much higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence. A ''T''<sub>m</sub> significantly lower than the annealing temperature may fail to anneal and extend at all.

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}}</ref> as do commercial software products such as ePrime and [[Beacon Designer]]. Computer simulations of theoretical PCR results ([[In silico PCR|Electronic PCR]]) may be performed to assist in primer design by giving melting and annealing temperatures, etc.<ref name="ePCR2">{{cite web|url=https://www.ncbi.nlm.nih.gov/sutils/e-pcr/|title=Electronic PCR|publisher=NCBI - National Center for Biotechnology Information|access-date=13 March 2012}}</ref>

As of 2014, many online tools are freely available for primer design, some of which focus on specific applications of PCR. Primers with high specificity for a subset of DNA templates in the presence of many similar variants can be designed using by some software (e.g. [[DECIPHER (software)|DECIPHER]]<ref name=About>{{cite web |url=https://decipher.sanger.ac.uk/about |title=About DECIPHER |publisher= Wellcome Trust Sanger Institute |access-date=12 February 2014}}</ref>) or be developed independently for a specific group of animals.<ref>{{Cite journal |last1=Karabanov |first1=D.P. |last2=Bekker |first2=E.I. |last3=Pavlov |first3=D.D. |last4=Borovikova |first4=E.A. |last5=Kodukhova |first5=Y.V. |last6=Kotov |first6=A.A. |date=1 February 2022 |title=New Sets of Primers for DNA Identification of Non-Indigenous Fish Species in the Volga-Kama Basin (European Russia) |journal=[[Water (journal)|Water]] |volume=14 |issue=3 |pages=437 |issn=2073-4441 |doi=10.3390/w14030437 |doi-access=free}}</ref>

Selecting a specific region of DNA for primer binding requires some additional considerations. Regions high in mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture; this phenomenon can lead to the production of 'primer dimer' products contaminating the end solution. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.

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>Adenosine added on the primer 50 end improved TA cloning efficiency of polymerase chain reaction products, Ri-He Peng, Ai-Sheng Xiong, Jin-ge Liu, Fang Xu, Cai Bin, Hong Zhu, Quan-Hong Yao</ref>

===Degenerate primers===
{{main|Degenerate bases}}
Some situations may call for the use of ''degenerate primers.'' These are mixtures of primers that are similar, but not identical. These may be convenient when amplifying the same [[gene]] from different [[organism]]s, as the sequences are probably similar but not identical. This technique is useful because the [[genetic code]] itself is [[Degenerate code|degenerate]], meaning several different [[codon]]s can code for the same [[amino acid]]. This allows different organisms to have a significantly different genetic sequence that code for a highly similar 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]], using the IUPAC symbols for [[degenerate bases]]. Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce the specificity of the PCR amplification.

''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 [[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.

==See also==
*[[Oligonucleotide synthesis]] &ndash; the methods by which primers are manufactured

==References==
{{Reflist}}

==External links==
* [http://frodo.wi.mit.edu/primer3/ Primer3]
* [https://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST]
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[[Category:DNA replication]]
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[[Category:Polymerase chain reaction]]