Does anyone have any experience with preferential allele amplification from PCR? I am trying to amplify over a troublesome repetitive region of which heterozygotes are proving difficult to call due to the wildtype allele amplifying preferentially over the mutant allele. I have already tried various PCR conditions and the best so far seems to be a PCR where the annealing and extension stage occurs simultaneously, however not all heterozygotes can be called and on agarose gel visualisation, the bands are so variable even when the starting DNA is normalised. If anyone has had any experience with this in the past it would be great to hear how you overcame this.
Thanks in advance!
Hi Rebekkah,
what kind of allele are you typing. Is it number of repetitions (how long is the repetitive sequence), or is it just SNP in close proximity of repetitive region? How do you type it?
Make sure, the sequence your primers anneal to is not polymorphic. Even simple SNP can damatically affect primer annealing. If such SNP is linked to your allele, it can easily lead to selective amplification.
Martin
if this is in a high CG repeat then melting may be an issue so I would try amplifying with high temperature annealing primers and in the presence of final concentration 1M betaine to assist the melting and slow the re-annealing of the template.It is a very cheap reagent or if anyone has Q solution from Promega I suspect they are the same thing
Hi Becky,
I got notified of this one. Am I correct that you have already tried a few non-overlapping primer sets? Have you chosen them such that products are the same length? If answers to both of these questions is yes, then we are looking at what is between them as the problem, rather than the primers themselves and I think Paul Rutland's advice is a good start. If that doesn't work, email me details of the primers and sequence.
David
Thank you all for your responses.
Martin - I should have mentioned the mutant allele is much longer than the wildtype allele and yes the region is very repetitive and actually very GT rich instead of GC rich. I don't think it is the primers that are the problem to be honest, I think it is the difficulty of what they are trying to amplify.
Paul - thank you for your suggestion, I will definitely give that a go. Is this similar to Q solution from Qiagen? Would this still work in a GT rich region? I tend to use this usually when I am struggling to amplify a GC rich region.
David - yes, I have already tried a couple of primer sets of similar length where the primers themselves do not overlap. I think you are right and it is the sequence that is being amplified that is the problem. I can give the betaine a go and see if this helps.
Thank you again!
yes Q solution is Qiagen not Promega..my mistake but it is a good product and works.
Self annealing can be a problem with high G sequences and a number of motifs will allow G quadruplex formation which may interfere with primer extension but quadruplexes are not as stable as correct base hydrogen bonding so given that the problem is with the longer allele I would make conditions as favourable as possible for the long allele production. These conditions ideally would be
1 long primers so as to ensure primer annealing before the template forms other structures.
2 Betaine/Q solution.
3 Increase the extension time...this will not have any effect on the short allele but may allow secondary structures to make and break and increase the efficiency of the amplification..
4 read up on similar applications such as pcr for triplet repeat expansion in disorders such as fragile x syndrome to see which enzymes are particularly suitable for long repeats ( I cannot help on this aspect )
As an after thought I wonder if nested pcr primers might give a good larger band...the smaller band would be too strong but if the size differece is large then the stronger smaller band will be far removed from the larger size band
second afterthought. Martins idea that one primer may have its 3' end covering a snp which is in linkage disequilibrium with the larger allele is very good and would certainly account for non amplification if the snp were anywhere in the end 4 bases of one primer
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