Are Purified Primers Really Necessary For Site-Directed Mutagenesis?

Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid templates.  Using purified primers is supposed to minimize the introduction of unintended mutations, thus drastically improving the probability of generating your desired mutant.  However, specially purified primers can be extremely expensive, and take longer to synthesize than standard primers.

In this article, we’ll discuss whether it’s necessary to use purified primers every time, or if there are some times when you can go with the cheaper, quicker option.

Why should you purify your oligos?

To understand why and how oligos are purified, you first need to understand a little bit about how they are synthesized.  Oligonucleotides are extended in a 3’ to 5’ direction from a solid support.  Bases are added individually to the growing chains until the sequence is complete, at which point the oligos are cleaved from the support and desalted for further applications.

During oligonucleotide synthesis, however, errors can occur, resulting in oligos with incorrect nucleotides, or shorter than the full sequence.  So, for example, if your primer as you designed it is 23 bases long, the final primer solution will contain oligos that are 22, 21, 20, or 19 bases pairs long, and so on.  For PCR amplification, this is not a big deal, since the majority of the oligonucleotides will be correct, and the 3’ end of the primer is the most important part for binding anyways.  However, for mutagenesis primers, the mismatched bases will be at the center of the oligo; thus, shorter versions of the oligo will not necessarily contain the appropriate site, and an unmutated copy of the plasmid will be amplified instead.

Types of oligonucleotide purification

There are two main types of purification for oligos that will be used for specialize applications:

  •  PAGE purification, or polyacrylamide gel electrophoresis, purifies full-length oligos based on size and conformation.  This technique is specifically recommended for oligos over 60 nucleotides, which are not as efficiently purified by HPLC as shorter oligos.  The drawback to PAGE purification is that products need to be extracted and desalted after purification, resulting in lower overall yields.
  • HPLC purification, or high performance liquid chromatography, purifies oligos by reverse-phase or anion-exchange chromatography.  This technique works especially well for purifying shorter oligos (around 15-25 base pairs), but is less efficient for longer strands.

When it may not be necessary

When considering whether or not to purify your primers, keep in mind what the goal of your experiment is.  For standard PCR amplification, there is rarely a need for purified primers, as discussed above.  If you’re using PCR to add a tag or linker to a DNA fragment, there is a chance that the oligo will be long enough to need purification – because, the longer the oligo, the more chances there are to generate an incorrect oligo.  When it comes to mutagenesis, it can be a tricky choice: unpurified oligos will not eliminate the possibility of getting a correctly mutated product, they will only decrease your chances.  I have successfully mutated plasmids using both purified and unpurified primers; of course, I have also failed to generate a mutant using both types of primers.  If you’ve introduced a restriction site along with your mutation, then screening by digest will eliminate the agony of sequencing clone upon clone looking for the right product; in this case, you may be able to get away with the lower yield of correct mutants from unpurified oligos.

At the end of the day, it comes down to a gamble: would you rather spend the money to increase your chances of getting a quick, accurate mutant, or try doing it cheaply and risk the possibility of having to start all over again if it doesn’t work?

What do you think – would you try mutagenesis with un-purified primers?

5 Comments

  1. mkc on July 27, 2012 at 5:58 am

    I always use standard desalted primers. I’ve never found purified primers to be necessary and in my humble opinion, they are not worth the extra cost and time.

  2. grahampositive on January 26, 2012 at 7:53 pm

    I have made hundreds of mutants using a modified SDM protocol. I have never used PAGE purified primers (for standard SDM- I have used them for ligation independent cloning). I always use the standard desalted invitrogen primers. My success rate is better than 75%, and I have never gotten incorrect mutations (results are always WT or desired mutant). The most important things I have found to be are: primer design and the TM of oligos vs annealing temp of reaction. I have found that if you take the time to carefully design your primers, you can achieve SDM at most sites with near 90% accuracy.

    • Radwan on November 15, 2013 at 6:04 pm

      Hi,
      Thanks for the information. What do you care about so much in designing the primers and if possible what do you recommend if i want to substitute 21 nucleotides in a 6 kb plasmid?
      Thanks again

  3. Roberto on January 6, 2012 at 4:01 pm

    Interesting. My point of view was that with such long primers as are used in SDM, a purity surely below 80% is to be expected for standard purifications – and if the mismatch is between the mutation site and the 3′, since Pfu will try to chew back a mismatched primer before extending it, the enzyme would eventually end up degrading the primer altogether instead of elongating it… thus reducing the efficiency of the PCR.
    So… I’ve always used PAGE-purified oligos. But I’m curious to read other people’s comments.

  4. James Lloyd on January 6, 2012 at 2:10 pm

    I have done only a limited amount of SDM but have never had a problem with using standard purification that couldn’t be blamed on something else. A few years ago I was trying to change a base to correct a mutation rather than redo the PCR to re-clone it. Every time I it corrected the problem but introduced a new insertion or deletion and always in the primer site. A colleague suggested the protein could be toxic to E. coli and once I took IPTG out of the blue-white selection I got the correct sequence with no new mutation.
    Clearly their was selection against this protein in E. coli and because primer synthesis is less accurate than Pfu in PCR the mutations I found were in the primer site. But greater purification would not have solved this problem. Only cost more money.

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