Hi Galt, Thanks, I'm aware how PCR primers work. But even Primer 3 only worries about the last 5 bases on the 3' end, not the last 15. For that matter, I just did a search for PCR Primer finding software, and the programs I found either only cared about the last 5 bases, defaulted to caring about less than that, or else didn't mention the 3' end at all.
For example, the PCR Primer Design Guidelines you suggested had this to say about the 3' end: 5. GC Clamp : The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer. 9. 3' End Stability : It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming. So, again, why do we need 15 bp perfect match at the 3' end of the primer, when no PCR Primer finding program worries about more than the last 5 bases? TIA, Greg ----- Original Message ----- From: "Galt Barber" <[email protected]> To: "Gregory Dougherty" <[email protected]> Cc: "genome" <[email protected]> Sent: Monday, August 2, 2010 1:01:16 PM GMT -06:00 US/Canada Central Subject: Re: [Genome] gfServer and gfPcr settings The 3' end of the primer is the one that will grow by Taq polymerase during the PCR reaction. It needs to be a perfect match for both specificity and to prime the reaction properly. A quick search found these pages: http://en.wikipedia.org/wiki/Polymerase_chain_reaction http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html On 08/02/10 09:51, Gregory Dougherty wrote: > I understand why gfServer requires a perfect match for tileSize + stepSize - > 1 bases. But when looking for primers, why does the perfect match have to be at the 3' end, rather than any place else in the primer? (Or is the usage information for gfPcr incorrect?) > > TIA, > > Greg > _______________________________________________ > Genome maillist - [email protected] > https://lists.soe.ucsc.edu/mailman/listinfo/genome _______________________________________________ Genome maillist - [email protected] https://lists.soe.ucsc.edu/mailman/listinfo/genome
