Roche's high throughput genotyping assay receives federal funding

Finding a SNP is only the first step. After that, comes the grunt work of determining its frequency in the population at large and in populations of people affected by a disease—a process requiring hundreds to thousands of genotype determinations. A technique under development at Roche Bioscience (Palo Alto, CA) called Kinetic PCR with Allele Specific Oligos has been given the nod by the National Human Genome Research Institute (NHGRI) for this application. The nod in this case is a $1.2-million, three-year grant to the developers of the technique Gary Peltz, head of genetics, Inflammatory Diseases Unit at Roche Bioscience, and Russell Higuchi, research leader in human genetics at Roche Molecular Systems (RMS).

Roche scientists are applying the new method to the study of the mouse genome, which is 80% homologous to the human genome, and for which a number of models for human disease exist (systemic lupus, emphasema, asthma, to name a few). Combining information from microarrays that compares expression profiles of normal and diseased animals with information on SNPs along mouse chromosomes, Roche is zeroing in on regions with potential for harboring disease-associated SNPs.

"The new process will enable scientists to rapidly scan the entire mouse genome for disease-associated risk factors. Experimental results can be extrapolated to identify genetic risk factors in the human genome," said Peltz. "In addition, the process has applications that are helpful for developing new diagnostic tools, targeted drug development, and in determining individual predisposition to certain diseases."

The new method, which according to Roche provides a faster and more cost-effective approach to identifying SNPs, has already yielded more than 300 murine SNP genotyping assays in less than six months, and is 30 to 50 times more efficient than current assay methods. It could become the industry standard for murine genotyping within the next several years.

Combining real-time PCR with allele-specific amplification, the assay can quantitate SNP frequency in pooled DNA samples. The pooled DNAs are subjected to two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. PCR product is quantitated in real time (no gel analysis is required) and by knowing the cycle number when the product goes above baseline, the allele frequency can be calculated. For example, for pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts.

In a paper in Genome Research published earlier this year, Roche scientists demonstrated that the assay can accurately determine SNP allele frequencies from 5% to 95%, using pools of both human and mouse DNAs that contained known quantities of eight different SNPs in total.

The new method offers two advantages over conventional technologies. It can be used for genotyping pooled DNA samples, which markedly reduces the number of genotyping assays that have to be performed. In addition, SNP-based polymorphisms will provide a much denser data set for analyzing DNA samples than microsatellite markers previously used for genotyping. Providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

Also collaborating on this project is Steven Shapiro of the Washington University-St. Louis, who is providing experimental models for emphysema.

The new methodology is expected to dramatically accelerate efforts to build a repository of mouse genetics data. "We've made great strides in developing mouse genotyping assays at RMS," said Higuchi. "I believe that using mouse models to identify genetic counterparts to human disease is the best way to proceed because it will shorten the time to testing human disease cohorts." Roche plans to apply this methodology initially to studying DNA samples obtained from individuals with osteoporosis and other conditions to identify genetic factors associated with these diseases.

Roche Bioscience scientists focus on the discovery and early clinical development of new medicines to treat diseases including arthritis, asthma, and other respiratory diseases; anxiety, depression, schizophrenia, and other neuropsychiatric diseases; genitourinary diseases; and osteoporosis.

For more information, contact Darien E. Wilson of Roche at 973-562-2232 or darien_e.wilson@roche.com.

Edited by Laura DeFrancesco
Managing Editor, Bioresearch Online
ldefrancesco@bioresearchonline.com

Source: Hoffman-La Roche Inc.