Primer design: Difference between revisions
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Primers are Watson-Crick complimentary short nucleotide sequences used to initiate DNA replication and amplification through PCR or to identify target sequences within DNA: | Primers are [[Watson-Crick_base_pairing|Watson-Crick]] complimentary short [[nucleotide|nucleotide]] sequences used to initiate [[DNA|DNA]] replication and amplification through [[PCR|PCR]] or to identify target sequences within DNA: | ||
*Ideally primers should be 18-20 base pairs in length, this is so that they are unique enough to anneal to the DNA at the flanking sequences of the fragment/gene we wish to amplify or isolate and so that they anneal to the DNA after it has been melted before the rest of the strands re-anneal. they are also kept at this length to ensure that they do not "mis-prime" if 1 out of the 20 bases does not bind then | *Ideally primers should be 18-20 base pairs in length, this is so that they are unique enough to anneal to the DNA at the flanking sequences of the fragment/gene we wish to amplify or isolate and so that they anneal to the DNA after it has been melted before the rest of the strands re-anneal. they are also kept at this length to ensure that they do not "mis-prime" if 1 out of the 20 bases does not bind then polymerase will not be able to synthesise new DNA." | ||
*Primers also cannot be complimentary to each other, if so these form "primer dimers". this is when they anneal and are amplified instead of the intended DNA sameple. | *Primers also cannot be complimentary to each other, if so these form "primer dimers". this is when they anneal and are amplified instead of the intended DNA sameple. | ||
*Primers can also contain "dangling ends" these are restriction endonuclease sites at the outside edges of the primers that enable the DNA to be amplified to be inserted into a plasmid or genome cut by the same restirction endonuclease. | *Primers can also contain "dangling ends" these are restriction endonuclease sites at the outside edges of the primers that enable the DNA to be amplified to be inserted into a plasmid or genome cut by the same restirction endonuclease. | ||
*Bases T and A should be avoided at the 3' end of the primer (as this is where polymerase binds) as they are more likely to mis-pair. G and C are much more stable. | *Bases T and A should be avoided at the 3' end of the primer (as this is where polymerase binds) as they are more likely to mis-pair. G and C are much more stable. | ||
*Primers can be modified to contain flourophores so that when they bind to target sequences of DNA we can visualise and isolate them etc. | *Primers can be modified to contain flourophores so that when they bind to target sequences of DNA we can visualise and isolate them etc. | ||
See also: | See also: | ||
[[PCR|PCR ]]<br>[[ | [[PCR|PCR ]]<br>[[Recombinant DNA Technology|Recombinant DNA technology]] | ||
Revision as of 15:54, 22 October 2012
Primers are Watson-Crick complimentary short nucleotide sequences used to initiate DNA replication and amplification through PCR or to identify target sequences within DNA:
- Ideally primers should be 18-20 base pairs in length, this is so that they are unique enough to anneal to the DNA at the flanking sequences of the fragment/gene we wish to amplify or isolate and so that they anneal to the DNA after it has been melted before the rest of the strands re-anneal. they are also kept at this length to ensure that they do not "mis-prime" if 1 out of the 20 bases does not bind then polymerase will not be able to synthesise new DNA."
- Primers also cannot be complimentary to each other, if so these form "primer dimers". this is when they anneal and are amplified instead of the intended DNA sameple.
- Primers can also contain "dangling ends" these are restriction endonuclease sites at the outside edges of the primers that enable the DNA to be amplified to be inserted into a plasmid or genome cut by the same restirction endonuclease.
- Bases T and A should be avoided at the 3' end of the primer (as this is where polymerase binds) as they are more likely to mis-pair. G and C are much more stable.
- Primers can be modified to contain flourophores so that when they bind to target sequences of DNA we can visualise and isolate them etc.
See also: