Dear fellow researchers.
I keep getting point mutations, most are single basepair delations, in my PCR products. All mutations occurred in the forward primer-annealing regions. Since there is no DNA elongation reaction in this region, I don't think the mutations have anything to do with DNA polymerase and elongation reaction. I am thinking the mutations were either from templates or primer. If the templates have no mutations, can I assume the primers we obtained commercially introduced those mutations? What else could cause the mutations? Thank you
Hi Rongfeng,
yes indeed, there are Companies that have very bad quality procedures or machines for the synthesis of oligonucleotide and the most common error is a missed incorporation of a nucleotide (the mutation cannot be in your template, since the amplified fragment starts with your primer: if it can anneal, which means that the skipped - or in some cases mutated - nucleotide is farther than 10-12 nt from the 3' end of the primer, you'll have it in your product).
As Ramachandramouli mentioned, HPLC purification will remove these shorter oligos, but the cost for the oligos will substantially increase. Choosing a Company that has good synthesis procedures will spare you money and allow anyway to have error-free primers. Here in Europe, we never had any problem with IDT, Sigma or Life Technologies oligos (also long ones up to 136 nt), while we had immense problems with Eurofins...
Good luck.
In my experience, the commercial primers which are purified by desalting method had same issues. The desalted primers normally contain deletions or additions. These are good for normal PCR applications. If you are using the primes for cloning, order HPLC purified, which have minimal deletions or additions. Good luck
Just to make sure I understood, are these mutations all with the same primer? Or are they happening with different primers, always in the forward (but not the reverse) position?
In the first case it's an oligo synthesis problem. Usually the mutations are always the same, but I've seen it happen in different positions. Probably issues with the synthesizer/incomplete deprotection. Take your case to your oligo supplier, they should provide a replacement oligo free of charge.
Hope it helps!
Hi Rongfeng,
yes indeed, there are Companies that have very bad quality procedures or machines for the synthesis of oligonucleotide and the most common error is a missed incorporation of a nucleotide (the mutation cannot be in your template, since the amplified fragment starts with your primer: if it can anneal, which means that the skipped - or in some cases mutated - nucleotide is farther than 10-12 nt from the 3' end of the primer, you'll have it in your product).
As Ramachandramouli mentioned, HPLC purification will remove these shorter oligos, but the cost for the oligos will substantially increase. Choosing a Company that has good synthesis procedures will spare you money and allow anyway to have error-free primers. Here in Europe, we never had any problem with IDT, Sigma or Life Technologies oligos (also long ones up to 136 nt), while we had immense problems with Eurofins...
Good luck.
Thank you all for the answers. To answer Alejandro Martin, these mutations are all from the same pair of primers. We have been ordering primers from the same company for years. This is the first time it happened, I guess we have been pretty lucky. I just confirmed that my template had no mutations, so the primer is the one to blame. Thank you all again for the help. You guys are the best.
Hi Rongfeng,
I think the discussion is almost closed and your item is solved but I would take your attention to another point. It was not really clear for me, why you are so interested in the primer binding sites derived from a sequenced PCR product. You will never get the natural sequence motif from the annealing region of a PCR product by sanger sequencing. This product is always let me say a "chimera" because the primers are added in excess and after a few cycles the motifs of the synthetic primers become manifest. Thats why exclusion of these regions in front of any downstream analysis is a MUST. On the other hand these "mutations" don't matter if the PCR is working well.
Sorry Dietmar, but you are quite out of topic. What you refer to is simple diagnostic PCR, for example for genotyping purposes, or to show the presence of a mRNA or not...
The case here is obviously different. PCR amplification is likely needed for cloning. Primers can contain restriction sites which MUST NOT be mutated in order to clone. Primers span start/stop codons if you are cloning cDNAs and they MUST NOT be mutated, as well as any other coding codon. Primer annealing regions MUST BE sequenced after any cloning using external sequencing primers or internal ones, just like Rongfeng did. They can be source of mutations just like the polymerase and one must make sure everything is fine after cloning.
Cheers
Dear Dietmar Lieckfeldt. The mutations were introduced by primers for cloning, not those for sequencing. When the PCR products were sequenced, we use m13F and m13R universal primers 400~ bp upstream of the beginning of PCR products. In other words, the mutations are inside the gene and cause problems for downstream work. Thank you
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