Development of a novel whole genome amplification method that mimics nature

DESCRIPTION (provided by applicant): BioHelix is the exclusive licensee for a novel, primase-based Whole Genome Amplification (pWGA) technology invented by Drs. Stanley Tabor and Charles Richardson at Harvard Medical School. This pWGA system utilizes multi ple replication proteins including a primase/helicase, a DNA polymerase, a single-stranded DNA binding protein, and several other accessory proteins to achieve rapid and sensitive whole genome amplification. As such, bringing this novel technology from the university laboratory to the market place is a challenging task. During Phase I, we evaluated the performance of this technology and successfully purified all necessary proteins in large production scale. We successfully launched the first generation Rapi some pWGA kit, which is, to our knowledge, the first commercially available DNA amplification system reconstructed from a cellular replisome. We have also discovered additional advantageous features of the pWGA platform. First, we have shown that the pWGA platform can perform real-time detection of trace amounts of DNA (100 fg). In addition, we have demonstrated that in comparison with another commercially available WGA product (GenomiPhi from GE Healthcare) the pWGA platform is particularly efficient in a mplifying (by 108 fold) circular DNA from low copy numbers (100 copies) of input. The overall goal of the Phase II research will be: 1) To continue to improve the performance of the pWGA system by optimizing the key components and fine-tuning each componen t (if successful, a second-generation pWGA kit with lower amplification bias and higher fidelity will be launched); 2) To explore new applications for pWGA in biomedical research and clinical diagnostics based on the aforementioned studies in Phase I. Base d on the phase I data, the applications of pWGA may be expanded into two new areas. One is as a universal DNA detection system, which will be developed for the detection of trace amounts of DNA contamination in biological samples or pharmaceutical products . The second new application is related to circular DNA amplification, based upon the phase I observation that pWGA can amplify circular DNA with extremely high efficiency (from 100 copies to over 1010). We propose to develop pWGA as a tool for clinical re search and diagnostic applications targeting circular DNA viruses such as human Papillomavirus (HPV). We will use pWGA to amplify samples containing HPV and subsequently determine the HPV genotype using the Luminex system. PUBLIC HEALTH RELEVANCE: Whole Ge nome Amplification (WGA) technologies are useful tools for cancer and genetic research. Amplifying the entire genome enables researchers to perform more tests on a given sample than would otherwise be possible. Two other types of WGA have been commercializ ed for research applications: 1) methods derived from the polymerase chain reaction and 2) multiple displacement amplification (MDA). Rubicon Genomics has developed a PCR-based WGA platform GenomePlex Kits for Research Use, available through Sigma-Aldrich . GE Healthcare markets the MDA technology under the name GenomiPhi while Qiagen sells MDA kits under the name REPLI-g(R). Both of these technologies have some limitations. The GenomePlex system generates short PCR amplicons, which limits its use in down stream applications such as RELP, DNA sequencing, and cloning. In addition, it requires a multiple-step reaction setup. MDA also often requires a heat-denaturation step before isothermal DNA amplification to facilitate primer annealing, which increases the complexity of the reaction setup and may introduce mutations into the template. In addition, the use of random primers in MDA makes it prone to non-specific DNA amplification even in the absence of an input template. The primase-base Whole Genome Amplific ation (pWGA) platform is an in vitro reconstruction of a cellular replisome. It performs a simple, one-step isothermal whole genome amplification without ad