K-072 Health Sciences
Research in the Loeb Lab is focused on the role of mutations in the initiation and progression of human cancers. These studies in the lab are centered on determining the accuracy of DNA synthesis by biochemical and genetic assays, or on measuring mutations in normal cells and in human tumors. We wish to identify the sources of spontaneous mutations and the role of mutations in driving tumor progression. Is the mutation rate in cancers greater than in normal cells and does increased mutation frequency drive the evolution of human tumors? Does increased mutation frequency allow tumors to divide where they ought not to, to invade, and to metastasize? Our current research efforts are in the following areas:
1) Human cancers express a mutator phenotype: For many years we have advanced the hypothesis that the mutation frequency in human tumors is greater than in corresponding normal cells- we proposed that cancers exhibit a mutator phenotype. The basic concept is that normal mutation rates are very low and insufficient to account for the large numbers of mutations in tumors. Until recently, this hypothesis was ignored. However, evidence by DNA sequencing now verifies that human tumors have thousands of clonal mutations. We have established several assays to measure rare random mutations and subclonal mutations in human tumors that are being used to address fundamental aspects of how cancers evolve.
2) Fidelity of DNA replication: We are continuing to study the mechanism(s) by which DNA polymerases can copy DNA with phenomenally high accuracy, approaching 1 error in every 10 million nucleotides polymerized. Our approach is to copy DNA in vitro with purified polymerases, transform bacteria with reaction products, and assay for the frequency and spectrum of mutations produced by the polymerase. An analysis of mutations produced by polymerases is key to evaluating the contribution of DNA replication to spontaneous mutations and to determine if reduced fidelity of DNA replication is causally associated with cancer.
3) Duplex DNA Sequencing: We have established a new method for DNA sequencing that offers unprecedented sensitivity and accuracy for detecting rare mutations in large population of heterogeneous DNAs. Our approach takes advantage of the complementary inherent in Watson-Crick DNA. Using linkers with complementary unique identifiers we can sequence each of two strands of a DNA duplex. True mutations would be present opposite each other and would be complementary.
Copyright © 2003-2014 Molecular & Cellular Biology Program, University of Washington
Fred Hutch | University of Washington
Institute for Systems Biology (ISB)| Center for Infectious Disease Research