change nothing. The annealing temperature of the primer in the first few cycles is determined only by the short region of perfect homology of the 3 prime end sequence of your primer  If the forward and reverse primers of the pcr have the same annealing temperature you still cannot increase the annealng temperature after the early cycles because then the other primer will not anneal. The primer concentration is in large excess so should not change and do not worry about the tail annealing as polymerases will not extend unless the 3' end of the oligo forms a double strand with the template . If the middle of your primer anneals there is no free end to extend from

change nothing. The annealing temperature of the primer in the first few cycles is determined only by the short region of perfect homology of the 3 prime end sequence of your primer  If the forward and reverse primers of the pcr have the same annealing temperature you still cannot increase the annealng temperature after the early cycles because then the other primer will not anneal. The primer concentration is in large excess so should not change and do not worry about the tail annealing as polymerases will not extend unless the 3' end of the oligo forms a double strand with the template . If the middle of your primer anneals there is no free end to extend from

Ditto. Crucially it is the last 5bp at the 3' end of the primer that are the most important

I cannot really add anything else to Pauls answer

Hello, at the practical level I agree, the PCR reaction starts with primers in excess, so unspecific binding of the 5' non-extensible ends (i.e. that do not anneal at the 3' end) will not affect greatly the PCR efficiency. However my guess is that as the reaction progresses and there is more template, non-specific (although non-productive) may affect the efficiency as there is less and less primer binding to the correct site. That's why I was suggesting a two-step PCR after the first few cycles.

Also, again not a problem at a practical level, Santalucia 2000 (http://nar.oxfordjournals.org/content/28/9/1929.short) note that in DNA probe design the dangling ends (short in this case) have a mostly stabilising effect, so my understanding is that in PCR, raising the annealing temperature would have a positive effect when applying the Ta determined for the non-tailed primer gives non-specific amplification.

Also, if non-specific binding of the 5' dangling end occurs in the middle of the amplicon, unless the polymerase has strand displacement I believe that correct amplification would be impaired and efficiency of the PCR would drop, so again doing two-step PCR could be beneficial.

Anyway, these are hypothetical considerations and in a standard PCR I would do as both of you suggest. If problems arise, I would try different approaches, which is actually the point of my question (which I can see I didn't ask straight, haha).

cheers,

Ruben

Still hypothetically, increasing primer concentration will consume your Mg ions (as the primers are more bigger) and make bining to sites (specific and unspecific) less stable.

And I thought, after few cycles, the binding will be more specific as there will be more colliding sites (and what the principle of stepdown pcr is).

Hello Ruben, because I was working with challenging GC-rich streptomycetes I used touchdown PCR (the primer had a 12bp long 5` dangling end). It´s related to the explanation Divya gave. In the first part of the PCR I start annealing at something like 2-3°C above the calculated annealing temperature to get more specific products and decreasing the temperature every cycle by 0.5°C for 10-13 cycles (worked well in my case). In the second phase you benefit from the presence of the specific products. Here you just run some additional PCR cycles with a decreased temperature (in my case 5-7°C less than the initial annealing temperature). Maybe that´s an option to try.

Best, Janine

Dear Ruben

This time I am in general agreement with Janine

If I have ambiguity where primer Tms are concerned then Touch down PCR where you transition from Tm +3C down to Tm-3C in 0.5C or 1c intervals per cycle followed by 25 x cycles at Tm-3C has worked well for me in the past, where standard 2 or 3 cycle standard PCR hasn't always worked. See for example some correspondence I had with a student regarding TD including an actual illustration of cycle design. Hope this helps !!

Find below a ref to principles of PCR including touch down PCR for difficult and/or non specific templates (pdf attached):

The protocol below which is one that I have successfully used many times assumes primer Tms of 59C and 60C respectively. I have given you Mike a word doc (which Maryam already has) for primer screening and Tm determination. Remember for accurate Tms you should include the Mg and dNTP conc in your reactions: During the initial Tm touch down phase you typically want to commence PCR at Tm + 3 = 63C and then step down in 0.5/1.0C increments until TM-3C  = ~ 57C. Then you build up product by standard amplification @ this Ta

http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html

95oC       3 min x1

95oC       15 sec

63oC*1    30 sec ( - 0.5oC/cycle)                                   x14

72oC       3 min

94oC       15 sec

57oC*2    30 sec                                x25

72oC       3 min

72oC       10 min   x1

4oC         ∞

*1: Initial Ta = Tm + 3oC

*2: Final Ta = Tm – 3oC

Hello,

Interesting comments! Actually I am struggling with overlapping PCR at the moment (and I believe that the problem is the junctions, because I have tried Gibson assembly of the modules and it does not work either, so I am redesigning the junctions), and for all the things that I have tried I had not thought of a touchdown. Thanks!

Cheers,

Ruben

it is the last 5bp at the 3' end of the primer that are the most important. on the other hand LAMP PCR technology could be looked at, a bulit in software will generate the primers and take are of the annealing temperature in regard to PCR amplicon amplification.

But, I saw change in efficiency of PCR up to 9 base(pretty significant change) from 3' end.

 https://www.researchgate.net/publication/233538525_Differential_haplotype_amplification_leads_to_misgenotyping_of_heterozygote_as_homozygote_when_using_single_nucleotide_mismatch_primer

Article Differential haplotype amplification leads to misgenotyping ...