Primer price crunch

Melinda / wikimedia.org

User:

Jose Royo, Andalusian Center for Development Biology, Spain

Project:

Determining the role of genetic variations in the development of human diseases and using this information to develop better drug targets

Problem:

Royo's group, then at Spanish biotech start-up Neocodex, had no SNP genotyping platform, so sequencing was affordable on the low scale, and he generally ran 5-15 SNPs in 1,000-2,000 samples. However, when medium-throughput projects arose, Royo's group faced the problem of genotyping 50-200 SNPs in 500 samples. "The investment in labeled oligos increased significantly the cost per sample," Royo writes in an E-mail, because they were faced with designing a different labeled primer for each SNP, the label being the most costly.

Solution:

Royo's group developed a "universal" labeled primer whose complementary sequence was incorporated as a 5ˊ tail of the specific primers (Nat Protoc, 2:1734-9, 2007). The protocol solves the problem and costs associated with labeling specific primers, he writes. The biotinylated primer incorporates into the specific PCR primer products, making it a "3-primer PCR," that leads right into the pyrosequencing steps and the SNP detection.

One limitation is that "you cannot say a priori how the 3-primer PCR will work and which stochiometry will be the best," Royo says. The stochiometry is tricky because the biotinylated primer sometimes interferes with the other primers, which can lower the PCR yield, so a researcher using this method would have to tweak the different concentrations. "There are other groups that proposed a similar strategy but using a different universal primer." There has been no comparison of the different strategies yet, he notes.

Cost:

Each biotinylated primer costs around $80 to $95, while a 40-mer oligo costs $20 to $30. Royo didn't have any platform costs to worry about, though.