Researchers at Stanford University have demonstrated yet another approach to single-molecule sequencing: They follow a single RNA polymerase enzyme and track its pauses as its travels along the DNA making RNA.
“It is an experiment that was really only a pipe dream many years ago,” says Steven Block, a professor of biological sciences and applied physics at Stanford. “What is truly new about this is we are using the motion — the mechanics of the enzyme — rather than its biochemistry to derive information.”
In their experiment, a proof-of-concept study, the researchers were able to identify correctly 30 out of 32 bases of a target sequence in less than three minutes of observation time using four DNA molecules.
“This is the smallest amount of DNA that has ever been used to sequence, so this is a record,” says Block, the senior investigator of the research, which appeared in Science in August.
Though he says that the speed, accuracy, read length, and throughput of the technique can be improved in principle, it is unclear whether it will have commercial applications, at least in the near term.
Block’s technology relies on two optical traps that each hold one polystyrene bead. One of the beads is attached to a molecule of E. coli RNA polymerase and the other to the end of a DNA molecule. As the enzyme proceeds along the DNA, making RNA, the distance between the two beads changes, which the scientists can measure with angstrom-level precision.
In order to read the bases, the researchers have to run the experiment four times, each time limiting one of the four nucleotides. Whenever the polymerase has to incorporate the limited nucleotide, it pauses for a brief moment. By analyzing the pauses in all four experiments, the scientists can deduce the entire sequence.
— Julia Karow
US Patent 7,094,531. Nucleic acid sequencing. Inventors: Gunter Schmidt and Andrew Hugin Thompson. Assignee: Xzillion. Issued: August 22, 2006. This patent covers a ligation method for sequencing DNA that consists of a cyclical process of probe hybridization, cleavable labeling, and ligation. The steps “are repeated in a cycle for a sufficient number of times to determine the sequence of … each single-stranded DNA by determining the sequence of release of each label,” according to the abstract.
US Patent 7,085,651. Method and device for assembling nucleic acid base sequences. Inventors: Tomohiro Yasuda and Tetsuo Nishikawa. Assignee: Hitachi. Issued: August 1, 2006. This patent relates to performing “the clustering and assembling of nucleic acid base sequences at a high speed,” according to its abstract. “If it is possible to assemble the sequences, they are assembled into a consensus sequence and also joined into the same cluster. The clustering and assembling are performed by repeatedly processing this procedure based on greedy method until no unprocessed input nucleic acid base sequence is left.”
The US Centers for Disease Control and Prevention released more than 650 genes from a number of flu viruses into GenBank, and expressed hopes that other governments would follow suit. The CDC’s step ensures that researchers will have access to sequence data from viruses isolated during the annual flu season in the US, from animal flu viruses that infect humans, and from novel strains that may emerge, including the H5N1 bird flu.
Researchers from the Virginia Bioinformatics Institute and the US Department of Energy’s Joint Genome Institute published the draft genome sequences of two plant pathogens, Phytophthora ramorum and Phytophthora sojae, which cause millions of dollars per year of damage to oak trees and soybean crops.
Applied Biosystems released the genetic analysis data file format and a data file converter for its CE-based sequencer to allow scientists and independent software vendors to develop bioinformatic applications targeted at its instruments. In other news, ABI settled its outstanding legal disputes with Promega over PCR technology.
A team of more than 50 scientists, led by Jonathan Eisen of the University of California, Davis, sequenced and analyzed the genome of the single-celled ciliate Tetrahymena thermophila. The organism, which has a micronucleus with five chromosomes and a macronucleus with more than 200 chromosomes, should give clues to eukaryotic evolution.
Cost to build New York’s new Forensic Biology DNA Laboratory, scheduled to be completed in November. The facility will contain more than 75 PCR instruments and sequencers from Applied Biosystems at a cost of some $5.5 million.