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Improved English, partially wikified, reduced degree of single-product hype. Article had focused on efficiency, neglected selectivity.
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The aim of '''in silico''' or digital or virtual or electronic [[PCR]] <ref>{{cite journal| author=Gregory D. Schuler |year=1997| journal=Genome Research| title=Sequence mapping by electronic PCR| pmid=9149949| volume=7| pages=541–550}}</ref> is to calculate theoretical PCR results by using [[DNA]] sequence(s) and [[Primer_(molecular_biology)|primers]] ([[Hybridization_probe|probes]]) <ref>{{cite journal| author=Kirill Rotmistrovsky, Wonhee Jang, Gregory D. Schuler |year=2004| journal=Nucleic Acids Research| title=
'''''In silico''''' '''PCR'''<ref>Synonyms: digital PCR, virtual PCR, electronic PCR, e-PCR</ref> refers to a computational procedure to calculate theoretical [[Polymerase chain reaction|polymerase chain reaction (PCR)]] results using a given set of [[Primer_(molecular_biology)|primers]] ([[Hybridization_probe|probes]]) to amplify [[DNA]] sequences from a sequenced [[genome]] or [[transcriptome]].<ref name=Schuler1997>{{cite pmid|9149949|noedit}}</ref><ref name=RotmistrovskyJang2004>{{cite pmid|15215361|noedit}}</ref><ref name=BikandiSanMilan2004>{{cite pmid|14752001|noedit}}</ref>
A web server for performing electronic PCR| pmid=15215361 | volume=32| pages=W108-W112| doi=10.1093/nar/gkh450}}</ref>, <ref>{{cite journal| author=Bikandi J, San Millan R, Rementeria A, Garaizar J |year=2004| journal=Bioinformatics| title=<i>In silico</i> analysis of complete bacterial genomes: PCR, AFLP-PCR, and endonuclease restriction| pmid=14752001 | volume=20| pages=798-799| doi=10.1093/bioinformatics/btg491}}</ref>.
The <i>in silico</i> tool is very attractive for quick analysing [[Primer_(molecular_biology)|primers]] or [[Hybridization_probe|probes]] through target [[DNA]] sequences, for determination primer or probe location, orientation, efficiency of binding, complementarity and [[DNA_melting|Tm]] calculation <ref>{{cite journal| author=Kalendar R, Lee D, Schulman AH |year=2011| journal=Genomics| title=Java web tools for PCR, <i>in silico</i> PCR, and oligonucleotide assembly and analysis|pmid=21569836|volume=98| issue=2| pages=137-144| doi=10.1016/j.ygeno.2011.04.009}}</ref>.


These tools are used to optimize the design of primers for target DNA or [[cDNA]] sequences. Primer optimization has two goals: efficiency and selectivity. Efficiency involves taking into account such factors as GC-content, efficiency of binding, complementarity, [[Nucleic acid secondary structure|secondary structure]], and [[DNA_melting|annealing and melting point (Tm)]]. Primer selectivity requires that the primer pairs not fortuitously bind to random sites other than the target of interest, nor should the primer pairs bind to conserved regions of a gene family. If the selectivity is poor, a set of primers will amplify multiple products besides the target of interest.<ref name=BoutrosOkey2004>{{cite pmid|15073008|noedit}}</ref>
The prediction appropriated short or long primer (probe) annealing site is only one way for PCR product prediction.


The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by ''in silico'' PCR tools may include determining primer location, orientation, length of each [[amplicon]], simulation of [[electrophoretic]] mobility, identification of [[open reading frames]], and links to other web resources.<ref name=KalendarLee2011>{{cite pmid|21569836|noedit}}</ref><ref name=YuZhang2011>{{cite pmid|21779992|noedit}}</ref>
[[FastPCR]] software or [http://primerdigital.com/tools/ Java Web Tools] allows simultaneous testing of single primer or a set of primers designed for multiplex target sequences. It performs a fast, gapless alignment to test the complementarity of the primers to the target sequences. Probable PCR products can be found for linear and circular templates using standard or inverse PCR as well as for multiplex PCR.


Many software packages are available offering differing balances of feature set, ease of use, efficiency, and cost.<ref name=FastPCR/><ref>{{cite web|title=Oligomer Web Tools|url=http://www.oligomer.fi/site/?lan=3&page_id=33|publisher=Oligomer Oy, Finland|accessdate=13 March 2012}}</ref><ref>{{cite web|title=Primer-BLAST: Finding primers specific to your PCR template|url=http://www.ncbi.nlm.nih.gov/tools/primer-blast/|publisher=NCBI - National Center for Biotechnology Information|accessdate=13 March 2012}}</ref><ref>{{cite web|title=UCSC Genome Bioinformatics|url=http://genome.ucsc.edu/|publisher=UCSC Genome Bioinformatics Group|accessdate=13 March 2012}}</ref> For example, [[FastPCR]],<ref name=FastPCR>{{cite web|title=FastPCR|url=http://primerdigital.com/fastpcr.html|publisher=PrimerDigital Ltd|accessdate=13 March 2012}}</ref> a commercial application, allows simultaneous testing of a single primer or a set of primers designed for multiplex target sequences. It performs a fast, gapless alignment to test the complementarity of the primers to the target sequences. Probable PCR products can be found for linear and circular templates using standard or [[inverse PCR]] as well as for [[multiplex PCR]].
⚫
Primer can bind many predicted sequences in template, but only sequences with no or few mismatches (1 or 2 depends from place and nucleotide) at 3’end of primer can be used for polymerase extension. The last 10-12 bases at 3’end of primer are sensitive to initiation of polymerase extension and general primer stability on binding template site. Single mismatch at these last 10 bases at 3’end of primer depends from the position and the structure can slightly reduced the primer binding and PCR efficiency.


⚫
A primer may bind to many predicted sequences, but only sequences with no or few mismatches (1 or 2, depending on location and nucleotide) at the 3'&nbsp;end of the primer can be used for polymerase extension. The last 10-12 bases at the 3'&nbsp;end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3'&nbsp;end of the primer depends its position and local structure, reducing the primer binding, selectivity, and PCR efficiency.<ref name=KalendarLee2011/><ref name=YuZhang2011/>


== Software ==
==External links==
* [http://www.finnzymes.com/java_applets/index.html FinnZymes Webtools for PCR and qPCR]
* [http://www.oligomer.fi/site/?lan=3&page_id=33 Oligomer Web Tools]
* [http://eu.idtdna.com/scitools/ IDT SciTools]
* [http://www.ncbi.nlm.nih.gov/tools/primer-blast/ NCBI/Primer-BLAST(Primer3)]
⚫
* [http://primerdigital.com/tools/ Webtools for PCR, qPCR, ''in silico'' PCR and oligonucleotides]
* [http://www.ncbi.nlm.nih.gov/projects/e-pcr/ Electronic PCR]
* [http://www.ncbi.nlm.nih.gov/projects/e-pcr/ Electronic PCR]
* [http://insilico.ehu.es/ <i>In silico</i> simulation of molecular biology experiments]
* [http://insilico.ehu.es/ ''In silico'' simulation of molecular biology experiments]

⚫
* [http://primerdigital.com/tools/ Webtools for PCR, qPCR, <i>in silico</i> PCR and oligonucleotides]
⚫
== References ==
⚫
{{reflist}}


[[Category:Nucleic acids]]
[[Category:Nucleic acids]]
[[Category:Bioinformatics]]
[[Category:Bioinformatics]]

== See also ==
* [[Polymerase chain reaction]]

⚫
== References ==
⚫
{{reflist}}

Revision as of 05:11, 13 March 2012

In silico PCR[1] refers to a computational procedure to calculate theoretical polymerase chain reaction (PCR) results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome.[2][3][4]

These tools are used to optimize the design of primers for target DNA or cDNA sequences. Primer optimization has two goals: efficiency and selectivity. Efficiency involves taking into account such factors as GC-content, efficiency of binding, complementarity, secondary structure, and annealing and melting point (Tm). Primer selectivity requires that the primer pairs not fortuitously bind to random sites other than the target of interest, nor should the primer pairs bind to conserved regions of a gene family. If the selectivity is poor, a set of primers will amplify multiple products besides the target of interest.[5]

The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by in silico PCR tools may include determining primer location, orientation, length of each amplicon, simulation of electrophoretic mobility, identification of open reading frames, and links to other web resources.[6][7]

Many software packages are available offering differing balances of feature set, ease of use, efficiency, and cost.[8][9][10][11] For example, FastPCR,[8] a commercial application, allows simultaneous testing of a single primer or a set of primers designed for multiplex target sequences. It performs a fast, gapless alignment to test the complementarity of the primers to the target sequences. Probable PCR products can be found for linear and circular templates using standard or inverse PCR as well as for multiplex PCR.

A primer may bind to many predicted sequences, but only sequences with no or few mismatches (1 or 2, depending on location and nucleotide) at the 3' end of the primer can be used for polymerase extension. The last 10-12 bases at the 3' end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3' end of the primer depends its position and local structure, reducing the primer binding, selectivity, and PCR efficiency.[6][7]

External links

References

  1. ^ Synonyms: digital PCR, virtual PCR, electronic PCR, e-PCR
  2. ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 9149949, please use {{cite journal}} with |pmid=9149949 instead.
  3. ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 15215361, please use {{cite journal}} with |pmid=15215361 instead.
  4. ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 14752001, please use {{cite journal}} with |pmid=14752001 instead.
  5. ^ Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 15073008, please use {{cite journal}} with |pmid=15073008 instead.
  6. ^ a b Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 21569836, please use {{cite journal}} with |pmid=21569836 instead.
  7. ^ a b Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 21779992, please use {{cite journal}} with |pmid=21779992 instead.
  8. ^ a b "FastPCR". PrimerDigital Ltd. Retrieved 13 March 2012.
  9. ^ "Oligomer Web Tools". Oligomer Oy, Finland. Retrieved 13 March 2012.
  10. ^ "Primer-BLAST: Finding primers specific to your PCR template". NCBI - National Center for Biotechnology Information. Retrieved 13 March 2012.
  11. ^ "UCSC Genome Bioinformatics". UCSC Genome Bioinformatics Group. Retrieved 13 March 2012.
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