Detection of specific DNA sequence through PCR

Dr N.I. Leaves nleaves at hgmp.mrc.ac.uk
Thu Sep 27 10:06:43 EST 2001


On 27 Sep 2001, darnel wrote:

> We will be amplifying a specific DNA sequence from isolated genomic DNA
> through PCR. Our goal is to determine the presence of that gene in another 
> frog specie. That specific DNA sequence is  known to be 120 bp long. If in
> our PCR experiment, we were able to amplify a fragment with nearly the same
> value (120 bp), how sure are we that we have really amplified and detected
> the same gene in another specie?? Thanks
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=3927&t=3927>
> 
I think your question can be broken down into two parts.

Firstly, how specific is a PCR product to a template? Assuming a random
sequence and two 20bp primers, then you get a specificity of 4^40 or a 1
in 1*10^24 assumming all 20 bases are required to anneal to get a
product. This is way in excess of the specificity needed in genomic
PCR. However, there are lots of wobble factors that you need to
consider and which actually make this figure a nonsense. On the negative
side you certainly dont need to get all 20 bases annealing to get a
product. Just annealing, say 5 bases, at the 3' end of both primers and a
low annealing temperature will dramatically reduce the specificity of the
rxn. On the positive side, not only do you need primers to anneal, they
need to be (in your example) ~120bp apart and be correctly directed. In
summary, seeing a single 120bp product and nothing else is probably very
informative.

Secondly, DNA is not a random sequence of nucleotides and it is
actually quite repititive. Amplifying coding DNA which is more specific is
more likely to produce a single product BUT by their nature genes occur in
families which often show very high similarity. Of course, seeing a
similar gene or the same gene across two species might just be a case of
semantics.

Practically, I would try the PCR and be pretty pleased with a clean
product of any size, and amazed by product of the same size. For
confirmation you might try digesting the product with a known restriction
enzyme or better still, sequence.

I hope my ramblings help. These are things which we all muse over and I'm
sure ther are lots of opinions out there.

Nick


end 
************************************************** 
Dr N I Leaves
Mouse Sequencing
MRC HGMP Resource Centre 
Hinxton 
Cambridge CB10 1SB 
tel: 01223 494557 (office) or 01223 494541 (lab)
email: nleaves at hgmp.mrc.ac.uk
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