Hi Galt, Thank you. I understand all that.
What I don't understand is why gfPCR requires that perfect match to be at the 3' end. Or, if it doesn't do that, I'd like to have that cleared up. IOW, I have a 22 base PCR Primer. Starting from the 5' end: if the first 15 bases match, then there's one base off, then the rest match, will gfPcr find that match? Assume, oh happy coincidence, that the primer is aligned with the tile "steps" so that the beginning of the primer falls on an indexed tile. TIA, Greg ----- Original Message ----- From: "Galt Barber" <[email protected]> To: "Gregory Dougherty" <[email protected]> Cc: "genome" <[email protected]> Sent: Monday, August 2, 2010 4:31:46 PM GMT -06:00 US/Canada Central Subject: Re: [Genome] gfServer and gfPcr settings The 15 minPerfect is a default value. hgPcr, isPcr, and gfPcr seem to tolerate setting it lower. This is because of the way BLAT works. This is the smallest size that blat can find an exact match for as you pointed out: tileSize + stepSize - 1 = 11 + 5 - 1 = 15 This provides the seed location. Then blat takes a block of dna around the seed. It searches for exact matches of size minPerfect in the block by direct memory compares. Then for each exact match it searches the remaining "good" match allowing 1 of out 3 bases to be substitutions (but no indels). So lowering the number of exact matches might pick up a little bit on sensitivity, but it will also tend to run slower as more fruitless leads are followed. If you have further suggestions for Jim Kent the author regarding BLAT PCR, you should contact him directly. If you need to tolerate more mismatches for very small sequences, perhaps a program like BWA, Bowtie, or SOAP2 would be useful. -Galt On 08/02/10 11:26, Gregory Dougherty wrote: > 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
