| The past decade has seen the emergence of nanopores as highly sensitive single molecule detectors. Recently, there has been interest in using this technique to rapidly and inexpensively sequence single molecules of DNA. In this process, DNA is electrophoretically driven through the nanopore bathed in an electrolyte solution, and the resultant fluctuations in the current carried by the nanopore are used to characterize the DNA. For DNA sequencing, each of the four nucleotides in DNA---adenine, thymine, cytosine, and guanine---must be detected individually and produce a current signals that are differentiable and identifiable. The primary focus of this thesis is to characterize and interpret blockade currents carried by the biological nanopore alpha-hemolysin (aHL) in the presence of single-stranded DNA (ssDNA). Examination of blockade currents produced by homopolymers of adenine, thymine and cytosine reveals the chemical orientation (3' leading or 5' leading) and identity of nucleotides of the homopolymer are important determinants of blockade current. In a follow up study, we find these current signals are highly sensitive to the identity of substituted nucleotides at multiple locations in an immobilized polythymine. Surprisingly, this sensitivity is neither a function of the geometry of the pore, nor the volume occupied by the substituted base. Blockade currents are in fact governed by base specific interactions between DNA and the aHL protein itself, a finding consistent with recent work on a mutant form of aHL. These results represent a significant contribution towards understanding the origins of blockade currents carried by aHL, and may prove useful in guiding the further development of nanopores for DNA analysis and sequencing. This thesis concludes with a discussion on sources of variability in the experimental system, their effects on currents measured in blocked (ib) and clear (io) pores, and the validity of eliminating variation in ib through normalization by io. |
