Making primers

http://allelebiotech.com/blogs/2011/04/making-primers/

We often hear that oligos (primers) are a simple product, and they should work every single time. From a producer’s point of view, oligos are arguably the most complicated products among common molecular biology reagents, at least in terms of the number of chemical steps required. DNA synthesis starts from the 3’ end to the 5’ end (opposite to DNA polymerases) on a solid support (e.g. CPG beads). For the addition of each base, the process begins by removing a protection group via “Deblocking”; then activating the last base for coupling with an “Activator”, adding the current nucleotide in the chemical form of a phosphoramidite (4 times in our protocol), blocking un-reacted openings with “Capping A and B solutions” (again 4 times each), forming bonds between bases by oxidization with Iodine, then looping all the way back to the beginning of the cycle. Many of these chemicals are either highly sensitive to moisture or have a short shelf life (can go bad any time).

Coming back from a seminar, sitting at the lab desk, you know you have a new idea and some cloning to do, and of course, it must be done tomorrow. The first thing you do is to send in some oligo sequences online to a local synthesis company late in the afternoon after looking at some maps and sequences. Around noon the next day, the oligos will be delivered in person to your hands. Most times everything will just work out fine as far as experiments involving primers are concerned; others you get stuck here and there along the way of cloning. Chances are you have run into problems with primers not giving PCR signals or clones with mistakes in the primer regions at some points in your research career. Even if this has only happened a few times, the memory, as well as the dissatisfaction and anger, can last for quite some time.

Between ordering and delivering, oligos are made overnight; they are then post-synthesis processed (requiring several hours starting in the early morning), OD’ed, and concentration adjusted. Given that the machine completes the run without any problems (power or computer related), and none of the chemicals run out, the best quick indicator of a good run is a color change from the protection group removed by Deblock at the last base. If there is visible amount of blocking group at the end of the synthesis, as reflected by an orange color from a Trityl group, it is likely that the synthesis was efficient till the end. Unfortunately, the efficiency of adding each base is never 100%–accumulations of missing or, at a lower percentage, mistaken bases will add up, especially in long oligos. Purification will remove some of the oligos with deletions, but not all of the bad oligos. MASS analysis will help determine the approximate percentage of bad oligos, but it will require time and cost not typically chosen by customers. It is our hope that understanding how oligos are made will help with more effective use of oligos when you order oligos, conduct experiments using oligos, or clone with oligos.

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