External Primers?

[methionine]

Hi all,
I need help understanding what the purpose of external primers in two-step deletion PCR is. I can understand why you'd need the "internal" primers -- they're complementary to one another and will allow the DNA to amplify with a deleted region... but I don't understand what the role of external primers are.
It is important that I understand this, otherwise I probably won't design very good external primers.

thanks,
-M

[drhamill]

Deletions can often be observed with a single round of PCR. If you have good genomic DNA, primers that amplify very specifically, and a deletion that is large enough to resolve on a gel, they can be seen. However, a two-step, or nested primer procedure is sometimes used to ensure specificity. I'm including below two links that describe the nested PCR procedure and its rationale (though not specifically for deletions). I'm also including a reference that describes the procedure used to generate deletions in C. elegans, a model organsim for which an international consortium of scientists are intending to "knock-out" each of the ~19,000 genes in this organism using a nested PCR approach. Hope this helps!

http://www.bio.davidson.edu/courses/gen ... edPCR.html
http://en.wikipedia.org/wiki/Nested_PCR

Edgley, M., D'Souza, A., Moulder, G., McKay, S., Shen, B., Gilchrist, E., Moerman, D., and Barstead, R. (2002). Improved detection of small deletions in complex pools of DNA. Nucleic Acids Res. 30, e52.

[methionine]

Thank you for the resources.
I just have one more question-- what's the big deal with having a C or a G at the 3' end of a primer? I know, the vague, general response is that it'll allow the polymerase to add the nucleotides... but why? How? If somebody could answer my question or point me in the right direction, I'd greatly appreciate it.

-M

[methionine]

Oh yes, and is there a difference between overlapping PCR and nested PCR?

[carolinethorn]

Hi M,

Its been a while. Glad it sound like things are progressing well for you.

People tend to prefer not to put a G or C at the 3' end of primers because the 3' end is the part where the polymerase will extend from so you want that to be as specific in binding to the target as possible and if the is an accidental binding you want it to release quickly - because when G and C bind there are more hydrogen bonds the energy it takes to break a faulty binding is greater. You especially want to make sure that your primer pair will not bind to each other (primer dimers) - so you don't want a G at te 3' end of one and C at the 3' end of the other.

Yes there is a difference between nested and overlapping PCR. Nested means that you have a large amplicon and then a smaller one within it. Usually its used to increase product volume and specificity - the large amplicon from the outside primers is amplified first then the small one is amplified from the product using the nested primers. (so in the simplest terms - large to small products)
Overlapping PCR is usually used to introduce changes in the target by having a mutation in the primer that becomes incorporated in the first smaller amplicon(s) and then the next round uses the outside primers to generate the larger amplicon but with the changes. (so in simplest terms small products to large products)

Its easier to see this as a diagram but i can't find one on the internet.

-Caroline

[methionine]

Hi,
Thanks for clearing that up for me.

I'm doing the overlapping PCR to do a deletion in the intron. From what I understand, the two primers anneal to part of the gene, and have attached to them a sequence that is complimentary to another short sequence... far away from where it has annealed to. I probably did a bad job of explaining that, but here is a picture I sketched out:



The dotted line represents the base pairs I want to delete, and the orange is complimentary, and the green is complimentary. I thought that half the primer would anneal to one area and "skip over" the area I wanted to delete...

I had tried to understand it from reading this:


Please correct me if any of my ideas are incorrect!


I'm a little confused because you said that people tend to NOT put a C or G at the 3' end of the primer, but I had looked it up online as well and I found:

3’-end Sequence

It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming(5). We have already explored the problem of primer homologies occurring at these regions. Another variable to look at is the inclusion of a G or C residue at the 3' end of primers. This “GC Clamp� helps to ensure correct binding at the 3' end due to the stronger hydrogen bonding of G/C residues. It also helps to improve the efficiency of the reaction by minimizing any “breathing� that might occur.

I know that G-C bonds have three hydrogen bonds instead of two in the A-T... But I'm not sure which one is correct now.

Thanks,
-M

[carolinethorn]

Hi M,

When designing primers there does tend to be a little voodoo. A good scientist can come up with explanations using the known facts (about G-C bonds and specificity at the 3' end) to explain why a given set of primers probably will or won't work but often there is a bit of trial and error, some tinkering with temperatures and so on! So I can see their point. Its not always practical though if you have to put the primer at a certain place or are trying to get the Tms of the 2 primers to match. Perhaps i overstated by saying "people tned not to" and should have just said that i have a preference to put C or G at the 3' end of one primer and A or T on the other. The main things though are still making sure the 3' end is going to bind to the target in the place you want it and not to the other primer.

Your diagram is a good way to explain. I am not clear though if the green and orange represent 2 first round products of PCR that will then be annealed to each other and amplifed together or if these are long overlapping primers. If they are first round products then you may have some issues with direction if i understand correctly what is labelled as 5' and 3'.

-Caroline

[methionine]

Yes, I did have trouble with the 5' and 3' ends... I know it's somehing along the lines of that, although I doubt I have it completely correct. I'm seeing my mentor tomorrow, so maybe I'll better understand how it all works out.

[carolinethorn]

That's good. I find it really helps to look at the real sequence and write out what you think the primers should be and check it again that way. Sometimes with just using lines you can't decide if they were complimentary or the same direction and then all the 5's and 3's get confued!

As always, i think you have enough background about how things are supposed to work out and what questions need to be resolved that you can have an intelligent discussion about it with your mentor, so I am confident you will work it out.

-Caroline