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Screening SNPs with Millions of Oligos

That's a Lot of Oligos!

Curtis Kautzer is the consummate IDT customer, having used IDT products in numerous applications through his career (primers for long range sequencing of the human genome, extra long oligos as PCR assay targets, adapters for DNA sequencing) and having placed one of the biggest all time orders—over $1 million worth of oligos! Currently working at Locus Development, Inc, he has molded himself as the “go-to” project leader for biotech companies that need help setting up new technologies.

SNP Map for Human Genome

Kautzer’s big oligo purchase was for primers—a hundred 96-well plates a week—which were used in a large genome project at Perlegen, a spin-off of Affymetrix. The research group resequenced the human genome of 25 individuals with the goal of generating a SNP map for the entire human genome. They initially performed long range PCR on 5−15 kb chunks of the genome, fragmented and labeled these, and hybridized the fragments to Affymetrix custom-designed chips. Results were compared to the reference sequence from the Human Genome Project to identify differences between the 25 individuals. The group then used short range PCR primers for 100−300 bp amplification around those areas. Eventually this work led to genome-wide association studies (GWAS) provided as a service for large pharmaceutical companies interested in genetic components of specific diseases.

“Because of my experience with IDT, I have never considered going elsewhere for oligo products, even after moving on from Perlegen to Navigenics and Locus Development”, notes Kautzer. “IDT is the only company I trust with my oligo orders. The local field representatives have had a great deal to do with this as they have been there immediately with solutions or recommendations during technical challenges, and have helped resolve any problems I have encountered.” 

Comparing Oligonucleotide Sources

The Perlegen scientists found over 1−2 million common SNPs, which were used to map mutations throughout the genome. The project lasted 3-4 years with primers ordered day in and day out—”…millions and millions of primers…”, says Kautzer. Because of the incredible volume of oligos his company consumed during this project, Krautzer compared different oligo vendors both in the US and abroad. He found that IDT was not only price competitive, but they were the most reliable and had the most consistent high quality. Furthermore, the quality of the next best source, Perlegen’s secondary or back up vendor, supplied oligos with a 10% lower success rate than that for the IDT oligos.”  Being able to avoid redesigning 50 primers out of 500 because they all work at IDT whereas they don’t somewhere else—that was critical to me.”

Long Oligo Mimics of Rare Mutations   

At Navigenics, Curtis Kautzer and his IDT field rep, Jeff Clark, designed 200mer (Ultramer) oligonucleotides that imitated rare homozygous mutations. These long oligos generated 3 different genotypes for 170 SNPs that, along with actual samples, defined the SNP clusters in the TaqMan assays used by the Navigenics production group. “Without the data points from the long oligos, the software used to autocall the different clusters has difficulty defining the SNPs properly; including these artificial targets allowed us to automate data analysis, removing the human error from the production line,” remarks Curtis.

Clinical Analysis Tools

Curtis and his colleagues at Locus Development, Inc are currently using the 454 Adaptors that IDT synthesizes. These adaptors are for ultra-deep amplicon sequencing on a Roche sequencer for detection of low frequency mutations. Locus Development uses this information to create bioinformatic tools that aid in the analysis of clinically relevant genetic markers.

Perlegen Project References
Patil N, Berno AJ, et al. (2001) Blocks of Limited Haplotype Diversity Revealed by High-Resolution Scanning of Human Chromosome 21. Science 294:1719−1723.

Frazer K, Chen X, et al. (2002) Genomic DNA Insertions and Deletions Occur Frequently Between Humans and Nonhuman Primates. Genome Res 13(3):341−346

Saccone SF, Hinrichs AL, et al. (2007) Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. Hum Mol Genet 16(1):36−49.


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