The potential use of nanopores for DNA sequencing has gained significant momentum. This is partly due to innovative solid state techniques, but more so due to breakthroughs using biological pores. One promise of nanopore sequencing has been very long read lengths, however we and others have performed most of our experiments using short synthetic DNA oligomers. In this proposal, we present experiments designed to test how efficiently nanopores can control and process long DNA templates (up to 2500 nt in length) as they are catalytically modified by DNA polymerases. Our work will focus on T7 DNA polymerase (T7 DNApol) and the Klenow fragment of DNA polymerase I (KF), coupled to the alpha hemolysin biopore (1-HL). There are three aims:
Aim 1. Limit DNA replication to template strands captured one-by-one in the nanopore. To ensure efficient serial analysis of individual DNA templates during catalysis, we will optimize a new strategy developed in our laboratory that quantitatively blocks DNA replication in bulk phase buffer bathing the nanopore, and that activates replication of individual DNA templates exclusively at the nanopore.
Aim 2. Quantify the effect of electrical force and DNA/pore interactions on DNA polymerase- dependent replication. Our objective is to determine the length of DNA template that can be reproducibly replicated on the nanopore. Three conditions (see figure below) will be examined to address three independent properties that could influence replication efficiency: a) Polymerase replication of long DNA templates captured in the nanopore under no load;b) Polymerase dependent replication of long DNA templates against resistive forces that arise from DNA/pore interactions;c) Polymerase dependent replication against a resistive electrical force.
Aim 3. Determine the effect of voltage on registry of large DNA templates in the nanopore at single nucleotide precision. Nanopore sequencing of intact DNA templates presupposes maintenance of single nucleotide spatial register during the time a base is read. For DNA-polymerase-controlled translocation this would be in the range of 1 to 100 milliseconds per measurement. At low voltages that are likely to permit polymerase catalysis, it is unclear if registry can be maintained.
High speed DNA sequencing is fundamental to understanding human diseases including cancer and heart disease. This proposal addresses fundamental questions about one promising new DNA sequencing technique based on biological nanopores.
| Cherf, Gerald M; Lieberman, Kate R; Rashid, Hytham et al. (2012) Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat Biotechnol 30:344-8 |
| Dahl, Joseph M; Mai, Ai H; Cherf, Gerald M et al. (2012) Direct observation of translocation in individual DNA polymerase complexes. J Biol Chem 287:13407-21 |
| Garalde, Daniel R; Simon, Christopher A; Dahl, Joseph M et al. (2011) Distinct complexes of DNA polymerase I (Klenow fragment) for base and sugar discrimination during nucleotide substrate selection. J Biol Chem 286:14480-92 |
| Olasagasti, Felix; Lieberman, Kate R; Benner, Seico et al. (2010) Replication of individual DNA molecules under electronic control using a protein nanopore. Nat Nanotechnol 5:798-806 |
| Lieberman, Kate R; Cherf, Gerald M; Doody, Michael J et al. (2010) Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J Am Chem Soc 132:17961-72 |
