Real-time DNA sequencing from single polymerase molecules

Methods Enzymol. 2010;472:431-55. doi: 10.1016/S0076-6879(10)72001-2.

Abstract

Pacific Biosciences has developed a method for real-time sequencing of single DNA molecules (Eid et al., 2009), with intrinsic sequencing rates of several bases per second and read lengths into the kilobase range. Conceptually, this sequencing approach is based on eavesdropping on the activity of DNA polymerase carrying out template-directed DNA polymerization. Performed in a highly parallel operational mode, sequential base additions catalyzed by each polymerase are detected with terminal phosphate-linked, fluorescence-labeled nucleotides. This chapter will first outline the principle of this single-molecule, real-time (SMRT) DNA sequencing method, followed by descriptions of its underlying components and typical sequencing run conditions. Two examples are provided which illustrate that, in addition to the DNA sequence, the dynamics of DNA polymerization from each enzyme molecules is directly accessible: the determination of base-specific kinetic parameters from single-molecule sequencing reads, and the characterization of DNA synthesis rate heterogeneities.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / metabolism*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Molecular Structure
  • Nucleotides / chemistry
  • Sequence Analysis, DNA / instrumentation
  • Sequence Analysis, DNA / methods*

Substances

  • Fluorescent Dyes
  • Nucleotides
  • DNA
  • DNA-Directed DNA Polymerase