Research Project
7328472
[unreadable] DESCRIPTION (provided by applicant): BioHelix is the exclusive licensee for the commercialization of 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 multiple replication proteins including a primase/helicase, a DNA polymerase, a single- stranded DNA binding protein, and several other accessory proteins. As such, bringing this novel technology from the university laboratory to the market place is a challenging task. The overall goal of the Phase I research is to determine the feasibility of commercializing pWGA technology for sale to researchers. One of the key aspects we propose to evaluate in the feasibility of commercially producing of all of the protein components used in pWGA. We will also evaluate the performance of pWGA, including reaction time, product yield, as well as product quality in terms of amplification bias using a combination of single nucleotide polymorphism (SNP) markers and microsatellite markers. Finally, we will evaluate the replication fidelity of the pWGA technology using genetic selection methods. The leading competitor for pWGA is multiple displacement amplification (MDA) marketed by GE Healthcare as GenomiPhi(tm) and Qiagen as REPLI-g(r). Studies based on highly informative microsatellite markers suggest MDA amplifications with small quantities of input DNA can result in a loss of genome coverage. If we can successfully commercialize pWGA, we may be able to offer researchers with a means of amplifying samples with very limited quantities of input DNA without loss of genome coverage. Indeed, pWGA uses a virtually complete replisome and thus may yield a more uniform amplification on entire genomes when limited numbers of initial copies are present. Moreover, the enzymes used by pWGA are also replicative polymerases, therefore we expect pWGA will have a fidelity of replication that is equivalent to MDA. Finally, the pWGA reaction appears much more rapid than MDA. Whole genome amplification technologies are a useful tool for cancer research and genetic research. Indeed, DNA samples used in these studies are often available in limited quantities. Amplifying the entire genome enables researchers to perform more tests on the samples than would otherwise be possible. Two other types of WGA are currently being commercialized for research applications: 1) methods derived from the polymerase chain reaction and 2) multiple displacement amplification (MDA). Rubicon Genomics commercializes GenomePlex(tm) Kits for Research Use through Sigma-Aldrich. GE Healthcare markets MDA technology under the name GenomiPhi(tm) while Qiagen sells MDA kits under the name REPLI-g(r). A major problem of the PCR-based methods is incomplete coverage of the genome due to amplification bias of PCR reactions over certain loci (e.g., GC rich regions). MDA is based on the strand displacement activity of phage Phi29 DNA polymerase, and uses random oligonucleotides of varying lengths (typically 6-mer to 8-mer) to prime DNA synthesis. MDA offers isothermal DNA amplification with less bias that PCR based methods. In addition, Phi29 DNA polymerase offers the hope of greater fidelity of replication than Taq DNA polymerase because it is a replicative polymerase. Despite this, studies based on highly informative microsatellite markers suggest MDA amplifications with small quantities of input DNA can result in a loss of genome coverage. The enzymes used by pWGA is also a replicative polymerase, therefore we expect pWGA will have a fidelity of replication that is equivalent to MDA. In addition, the pWGA reaction appears much more rapid than MDA. At this stage, we believe pWGA is at least equivalent to MDA in terms of amplification bias. The results we would obtain during this Phase I study will help determine if pWGA has less bias when small quantities of template DNA is used. [unreadable] [unreadable] [unreadable]
