Polynucleotide sequencing is the process of determining the nucleotide order of a given DNA or RNA fragment. These methods were first used in the sequencing of fragments, then individual genes, and have now been applied to whole genomes, from organisms as diverse as humans and E. coli. The desire for new sequence information has not been quenched by elucidation of these genomic sequences, however, as it has become apparent that individual variations in gene sequences play important roles in many physiological processes.
At the present time, most large scale sequencing efforts use the “chain termination”, or Sanger method. In this method, polymerization of DNA is initiated at a specific site on a template by using a short oligonucleotide primer. The primer is extended with DNA polymerase in the presence of four deoxynucleotides, along with a low concentration of a chain terminating nucleotide, for example a dideoxynucleotide. Limited incorporation of the chain terminating nucleotide results in a set of overlapping DNA fragments that are all terminated at a position corresponding to the dideoxynucleotide. The fragments are then size-separated by electrophoresis, e.g. by PAGE, capillary electrophoresis, etc. Detection of the fragments can utilize radioactive or fluorescent tags, and variations of the method use dye-labeled primers, dye-labeled terminator nucleotides, and the like.
Other methods are known in the art that utilize size fractionation of fragments, for example the Maxam-Gilbert method, in which chemical reactions that selectively cleave DNA are used to generate the set of fragments. In addition, bulk techniques have been proposed that do not utilize sizing of fragments, including sequencing by hybridization, in which an array of short sequences of nucleotide probes is brought in contact with the target DNA sequence. A biochemical method determines the subset of probes that bind to the target sequence (the spectrum of the sequence), and a combinatorial method is used to reconstruct the DNA sequence from the spectrum.
There have been many proposals to develop new sequencing technologies based on single molecule measurements, for example by observing the interaction of particular proteins with DNA or by using ultra high-resolution scanned probe microscopy. Unlike conventional technology, their speed and read length would not be limited by the resolving power of electrophoretic separation. Single molecule sensitivity might permit direct sequencing of mRNA from rare cell populations or individual cells. A major obstacle has been the high data density of DNA. Scanned probe microscopes have not yet been able to demonstrate simultaneously the resolution and chemical specificity needed to resolve individual bases.
Braslavsky et al. (2003) PNAS 100:3960-3964 imaged sequence information from a single DNA template as its complementary strand was synthesized. DNA template oligonucleotides were hybridized to a fluorescently labeled primer and bound to a solid surface via streptavidin and biotin with a surface density low enough to resolve single molecules. The primed templates were detected through their fluorescent tags, their locations were recorded for future reference, and the tags were photobleached. A combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfer was used to reduce background noise. Labeled nucleotide triphosphates and DNA polymerase enzyme were then washed in and out of the flow cell while the known locations of the DNA templates were monitored for the appearance of fluorescence. It was shown that DNA polymerase was active on surface-immobilized DNA templates and can incorporate nucleotides with high fidelity, although only a few nucleotide residues were actually sequenced. A disadvantage of this method is that it used information from many single molecule images to generate an ensemble measurement from which sequence information was extracted. Therefore information from many molecules was averaged together to obtain a single measurement.
An alternative method has been proposed (Akeson et al. (1999) Biophys J. 77(6):3227-33) in which single molecules of a polynucleotide are driven through a nanopore channel by an applied electric field. During translocation, nucleotides within the polynucleotide are reported to pass through the pore in sequential, single-file order because of the limiting diameter of the pore. It has been suggested that this passage could provide a means for discrimination between pyrimidine and purine segments. At this time however, nanopore sequencing is a theoretical method, with only limited lab bench results.
Methods of obtaining nucleotide sequences from small samples, down to single molecules, are of great interest for research, environmental and clinical purposes. The ability of such a method to provide information from a single molecule in the absence of averaging is desirable. The present invention addresses this issue.
Publications
Abbondanzieri et al. (2005) Nature 438:460-465 demonstrate direct observation of base pair stepping by RNA polymerase. Greenleaf et al. (2005) Phys. Rev. Lett. 95:208102 describe a passive all-optical force clamp for high-resolution laser trapping.