Rapid purification following enzyme-catalyzed nucleic acid reactions

DESCRIPTION (provided by applicant): Biochemical reactions used in the analysis and manipulation of DNA are important for many applications including forensics, diagnostic genetic testing and biomedical research. Enzymatic reactions to cut and insert DNA into cloning sites underpin many strategies for disease research and for use of microorganisms to express important proteins with medical value. Purification to obtain the desired reaction components while discarding those that can interfere with further use of the DNA is essential and comprises a significant contribution to the time, cost and labor involved in genomics. Improving workflow and enabling automation in the cleanup steps during library generation for next generation sequencing would remove one of the bottlenecks in that exciting and promising technology for genomic mapping. Diffinity Genomics has licensed novel materials technology developed in the PI's lab at the University of Rochester that can be used for fast, inexpensive and simple biomolecular separations needed to purify nucleic acid reactions. In particular, Diffinity has released a product enabling rapid, efficient purification of DNA after polymerase chain amplification prior to sequencing reactions. The work in the present proposal involves adapting that technology to make products for rapid purification of enzymatic reactions. Current approaches to purifying these reactions require multiple steps and use reagents to bind all of the biomolecules in solution to a substrate and then selectively redissolve the desired component. Diffinity's purification method is distinct because it uses specially configured surfaces that attract undesired components of a solution while leaving the desired ones in solution. This enables a single-step processthat can be implemented by retaining particles with the specially functionalized surfaces in pipette tips so that the purification process is reduced simply to aspirating the reaction solution and dispensing the purified DNA reaction solution. We will demonstrate that we can configure silica particle surfaces appropriate to one-step, 60 second extraction of the undesired components following enzymatic reactions while leaving the desired ones in solution. PUBLIC HEALTH RELEVANCE: Enzyme-catalyzed DNAreactions are widely used in health care applications such as diagnosis, pathogen detection and cloning for therapeutic purposes. These reactions routinely require purification that is costly and labor-intensive. We propose to make functional pipette tipscontaining novel filtration materials that can be used to dramatically reduce the time, cost and environmental waste associated with current purification protocols.