Rapid and Efficient PCR Cleanup Filters

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DESCRIPTION (provided by applicant): Analysis of DNA is important in many applications including forensics, agriculture, pathogen detection, diagnostic genetic testing and biomedical research. For the vast majority of genomic DNA analysis, chemical amplification methods such as polymerase chain reaction (PCR) amplification of a target sequence are used to increase the amount of the genetic fragment under study so that the sensitivity of common analytical methods is adequate. PCR, however, is only an early step for some important genomic assays such as capillary sequencing, MALDI-TOF based genotyping, microarray spotting, restriction analysis and cloning. For these, "clean up" of the PCR product is generally required, specifically to remove unamplified primers, excess nucleotides (dNTP) and additional organic components other than the desired amplicons. Cleanup products and protocols on the market are a significant contribution to the time, cost and labor involved in DNA analysis. They also exhibit poor recovery of DNA for relatively short amplicons (< 300 base pairs) as are preferred for speed of PCR and for a variety of applications such as multiplex PCR and handling of damaged DNA or RNA. Diffinity Genomics has licensed technology developed in the lab of the PI at the University of Rochester that is promising for single-step, 5 minute clean up of PCR product with high throughput for amplicons and efficient removal of primers and dNTP. The method has particular promise for the case of short amplicons cited above. The separation process exploits the recent finding that gold nanoparticles immobilized on high surface area substrates can selectively adsorb short, singlestranded DNA. Our goal is to take this observation and to produce a commercializable prototype kit suitable for the applications enumerated above. We will quantify the throughput of amplicons, primers and dNTP. Specific improvements to the form factor of the filter and surface attachment chemistry used to immobilize the gold nanoparticles will be evaluated for their effectiveness in increasing amplicon throughput and decreasing primer and dNTP throughput. We plan to increase surface area of silica beads utilized in the filters and the loading of gold nanoparticles in order to make the filters faster and longer lasting. We also propose to investigate uncharged mercaptosilane and negatively charged mixed monolayer coatings of the silica beads to reduce loss of amplicons in the filter. Improved recovery of the desired amplified DNA by mechanical removal of the beads will also be implemented. Finally, we have arranged for beta testing of the filter prototypes by prospective customers. Individual genetics will increasingly be used both to prescribe therapeutic courses and to develop personalized drugs so that screening and sequencing DNA are very important to medical research and diagnosis. Polymerase chain reaction (PCR) products are routinely used in DNA analysis but require lengthy and cumbersome cleanup prior to sequencing. We propose to commercialize filters that improve recovery of short DNA segments from PCR products as as reduce the time, cost and labor associated with the cleanup procedure.