Touchdown PCR question - why does this work?

[DacotahM]

I'm working on an arthropod similar to a hoverfly. Found a protocol for degenerate primers. It seems pretty odd, and it has a very cold annealing temperature that only gets colder, but we get product from primers that haven't worked before. I'd like to know why it seems to work. The program is way out of range for all the annealing temperatures of all the primers. I often run it twice using the first-round product as my template.

94 - Hold
94 - 5 min

CYCLE 24x
94 - 30"
50 - 30" - 0.4C per cycle
72 - 30" + 2" per cycle

CYCLE 12x
94 - 30"
40 - 40"
72 - 60" + 3" per cycle

72 - 5'
4 - Hold

[Irideus]

The calculated Tm is an approximation of the REAL Tm for the primers. There´s for example the classical 2(A+T)+4(G+C) method, the "salt adjusted" one, the "nearest neighbor", and so on. If there were an ultimate way of estimating the Tm, then we all would use a single formula.

Sometimes, primers that won´t anneal in the vecinity of their Tm do when the temperature is lowered enough.

The touchdown procedure allows better specificity in your PCR lag phase, by allowing very few primer molecules to anneal to your template, and assuring that annealing is specific. With cycles going by, the temperature is dropped stepwise in order to get more and more primer molecules to give off product, provided that your target is now abundant enough to neglect off-target mispriming.

But what is strange on your PCR (at least for me) is the increasing elongation times. It looks like your product would be getting longer with every cycle...(?)

Kindest regards.

Irideus

[jonoave]

Originally Posted by Irideus

But what is strange on your PCR (at least for me) is the increasing elongation times. It looks like your product would be getting longer with every cycle...(?)

Kindest regards.

Irideus

I agree with what Irideus said about Touchdown PCR; that's the basic concept of it. Perhaps the OP can read more on this concept.

As for the increasing elongation time, this is a suggested strategy for long-distance PCR. Supposedly it has something to do with the PCR reaction getting less efficient and/or the exonuclease activity of proofreading-polymerases (e.g. Pfu) taking up more time.

[LCMaul]

Saw this and the coincidence is that its running behind me right now. The reason one would run a touchdown PCR, besides the technique sounding as nearly cool as the Sonic Hedgehog gene, is that if you're having trouble figuring out the correct annealing temperature of your primer, this is a way to use primer competition to find the right annealing temperature.

The reason it starts out high is because it tries to find the annealing temperature of your primers and target first before it finds background. By starting high, once it gets to the actual unknown annealing temperature, it will then start producing target product . Using the principle of competitive resources, by the time it gets to the lower unspecific product, the target, which is now in high concentration, will outcompete other product and will be the dominant product.

In regard to primer that hasn't worked before, it could be you were using the wrong annealing temperature in the past.

The con ? Well, its not nearly as clean as if you knew your specific annealing temperature, but having something from nothing is a heck of a lot better !

[gautama]

Hi, sorry to bother you all.
I need some help with PCR and DGGE.

I am looking at the soil microbiota as a whole rather than isolating specific bacterial species. In my DGGE profile the intensities of each band is about the same at each lane
(seen at the same row), that's what make my head aches. I was hoping to see a different profile lane to lane, but it seems they were all look the same! But from one band to another in the same lane/column have different intensities. I used 338F-GC and 518R. Any ideas how can I avoid two bacterial species from stopping at the same point in my DGGE gel beside using different primers?
Thanks for your kind answer...

btw, i already make thread at
PCR to DGGE questions