There are many manufacturers of oligonucleotides and most supply good quality oligos. However, I have experienced differences in quality between manufacturers, especially with labeled oligos. Labeling efficiencies can vary between manufacturers and also between batches. In addition, not all manufacturers can supply you with exactly the modification you are looking for. I use many labeled oligos for my research and in hopes of maintaining satisfying results, I started to test our three main suppliers. I started by testing oligos labeled with fluorescent dyes (such as FAM, HEX and TET) for sequencing and also looked at the labeling efficiency of manufactured biotin-labeled primers/probes. I compared sequencing primers from Eurogentec, Applied Biosystems and Invitrogen. Since biotin-labeling is not available from Applied Biosystems, biotin-oligos were compared only between Eurogentec and Invitrogen.
I use the sequencing primers for the detection and easy scoring of microsatellites of the flea beetle Phyllotreta nemorum. We scored more than one thousand individuals with 10 loci each, using several batches of labeled primers and found no major differences between batches of sequencing primers from the same supplier or between different suppliers.
In our laboratory, we use a biotin-labeled primer in PCR for the detection of Borrelia infection in Ticks (Ixodus ricinus). The resulting PCR products are then used in a reverse line blot technique to distinguish between different Borrelia species. We have several years of experience with this technique. When all parameters are optimal, we can detect a few bacterial cells in a single tick. PCR reactions were optimized using the HotStarTaq Master Mix (cat. no. 203445) from Qiagen. The C6-amino-linked probes are always from the same supplier (Invitrogen). Positive, chemiluminescent reactions are detected using a peroxidase reaction kit (ECL Detection Reagents from GE Healthcare, cat. no. RPN2105) and light sensitive film. The Eurogentec biotin-labeled oligos always performed well with the same bright positive spots resulting from a high Borrelia infection rate. The results with the biotin-labeled oligos from Invitrogen, however, were not always consistent; results varied depending on the batch of oligos. This variation could lower the found infection rate by 5-7%, a significant amount considering that normal infection rates are between 7-17%, depending on sample sites. I think that this difference is due to a difference in the labeling efficiency when generating the biotin-labeled probes. Based on our results, we decided to use only Eurogentec biotin-labeled oligos.
Inefficiently labeled oligos is not the first thing one thinks of when looking to optimize an experimental setup. But our results clearly show that testing the labeled oligo is as important as optimizing other experimental parameters. I recommend trying different manufactures and using controls to test for consistent oligo labeling. One other (minor) point is the ease by which you can order oligos via Invitrogen’s website. Their website ordering system is much more intuitive and straightforward than that of their competitors.
Ordering Oligos: A comparison between oligonucleotides from Eurogentec, Applied Biosystems and Invitrogen
No difference was observed between sequencing primers from the three tested manufactures, but biotin-labeled oligos from Eurogentec were clearly superior to Invitrogen’s.
Labeling efficiency can vary and, therefore, is another parameter which should be tested and controlled.
The Bottom Line
It is important to test oligos prior to use. Biotin-labeled oligos of Eurogentec are superior to Invitrogen’s.