9 replies to this topic

[Adrian K]

Dear all,

I do have a strange encounter and wish to ask your opinion.

I had designed a 2 sets of primers where there are one mismatch on 3' (A on primer, G on template).

5'   xxxxxxxxxxxxxxxxA 3'
DNA  xxxxxxxxxxxxxxxxG

I have an impression that Taq polymerase will not going to extend in this situation and there will never be any pcr product amplified. However, one of the primer still yield amplification, even with the addition of 5% DMSO, gives very significant bright band as well. I'm quite puzzled.  

Any idea? Or is just my misconception?

Thanks.

Adrian

[phage434]

This is largely true, although rare extensions can occur.  Once one happens, then the PCR will amplify the single extended molecule, so the negative selection needs to be extremely strong.

A/C or G/T mismatches are pretty easy to make mistakes on.  C/T, C/C, T/T mismatches are also pretty easy.  The mismatch you want, if you can find one, is an A/A or A/G or G/G mismatch at the 3' end.  These will be relatively rare, since mutations also tend to do A<->G and C<->T substitutions.

High annealing temperatures will likely help in making the selection stronger.

[Adrian K]

Hi phage434,

Many thanks for your prompt reply. Thank you for the information which enlighten me a lot.
Many thanks again.

Adrian

[Trof]

Yes, one nucleotide mismatch may not be enough. Two is better. Did you try increasing the temperature up to the point were mismatch wouldn't be amplified and match will? Sometimes temperature difference between melting of match and mismatch can be very narrow, you can try LNA spiked primers that have higher temperature differences.

[Adrian K]

Thanks Trof for your suggestion. Honestly, this is my first time heard about LNA spiked primers. Where can I get more information about this primers? I try to google it but only shows somthing about real-time PCR..

please bear with my total ignorance...  

[Trof]

LNA "locked nuleic acid" is a nucleotide modification, that allows better single-base discrimination. Here is some basics in article of some Roche product.

Primary it was a invention of Exiqon, who is now seling patents (or how is that called) to other manufacturers (if you order oligo from someone else they pay to Exiqon, but Exiqon is not really cheap either). Since they're very good in distinguishing small mismatches, they have wide usage, in hybridisation, microRNAs, siRNA, everywhere including real-time PCR probes (that is the basis of Roche's Universal Probe Library mentioned in the link above). You can browse their SNP detection site to learn more.
We have real-time allelic discrimination probes and the UPL with LNA for some years now, works well.

[Adrian K]

Hi Trof, many thanks for the info.

[Adrian K]

phage434, on 12 July 2011 - 09:16 AM, said:

This is largely true, although rare extensions can occur.  Once one happens, then the PCR will amplify the single extended molecule, so the negative selection needs to be extremely strong.

A/C or G/T mismatches are pretty easy to make mistakes on.  C/T, C/C, T/T mismatches are also pretty easy.  The mismatch you want, if you can find one, is an A/A or A/G or G/G mismatch at the 3' end.  These will be relatively rare, since mutations also tend to do A<->G and C<->T substitutions.

High annealing temperatures will likely help in making the selection stronger.

Dear phage434,
Where can I find for more information for the above statement?
Many thanks.

[phage434]

I don't know.  The issue is physical interference.  The purine nucleotides (A, G) are bigger than the pyrimidines.  Two purines don't fit into the double helix easily, while a pair of pyrimidines, although mismatched, will.

[Adrian K]

I see I see... wow... physical interference, something I never thought of all these while...

Many thanks for the input. Cheers...