Journal: Nucleic Acids Research

Article Title: An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase–DNA complexes to surfaces

doi: 10.1093/nar/gkn531

Figure Lengend Snippet: Polymerization kinetics of purified complexes (see Materials and Methods section). DNA-synthesis rates ( v , nucleotides per second) were determined for various nucleotide substrate concentrations ( s , micromolar). ( A ) Purified complexes: K m = 54 μM, V max = 3.4 nt/s. ( B ) Control sample of uncomplexed polymerase and DNA: K m = 21 μM, V max = 12.0 nt/s.

Article Snippet: To obtain long single-molecule reads, potentially tens of kilobases, we and others ( ) ( http://www.pacificbiosciences.com , http://visigenbio.com ) are developing sequencing-by-synthesis approaches using phosphate-labeled nucleotides.

Techniques: Purification, DNA Synthesis

Journal: Nucleic Acids Research

Article Title: An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase–DNA complexes to surfaces

doi: 10.1093/nar/gkn531

Figure Lengend Snippet: DNA synthesis by immobilized complexes. Purified ternary complexes made with unlabeled streptavidin were immobilized in a reaction chamber on a PEG–biotin coated coverglass (see Materials and methods section). ( A ) The reaction chamber was filled with 1 μl of buffer C containing 5 mM MgCl2, 100 μM each of dATP, dCTP, dGTP and base-labeled Alexa Fluor-488-dUTP (Invitrogen). The chamber was sealed with a plastic coverslip and incubated in a humid jar at 54°C for 90 min. The chamber was rinsed with water to remove unincorporated nucleotides and the coverglass surface was imaged by TIRF microscopy (see Materials and methods section). The labeled complexes were seen waving back and forth under Brownian motion, while remaining tethered to the surface ( Supplementary Movie M1 ). ( B ) Control reaction inhibiting polymerase activity by replacing Mg++ with 0.1 mM EDTA. ( C ) Zoomed-in view of a single DNA spot from Movie M1 showing movement of about 1 μm leftward occurring between frames 61 and 62. A pixel is marked for reference ( x – y coordinate 386, 534). Exposure time was 80 ms and the pixel dimension 0.27 μ. Movie M1 was acquired in a replicate experiment as in (A).

Article Snippet: To obtain long single-molecule reads, potentially tens of kilobases, we and others ( ) ( http://www.pacificbiosciences.com , http://visigenbio.com ) are developing sequencing-by-synthesis approaches using phosphate-labeled nucleotides.

Techniques: DNA Synthesis, Purification, Labeling, Incubation, Microscopy, Activity Assay

Journal: Genome Biology

Article Title: The properties and applications of single-molecule DNA sequencing

doi: 10.1186/gb-2011-12-2-217

Figure Lengend Snippet: Sequence database submissions from 1982 to 2010 . Nucleotides submitted to the classical version of GenBank (diamonds, thin line) and to the Sequence Read Archive (circles, thick line) are shown as a function of time. Data for GenBank up to 2008 were obtained from the NCBI website and subsequent years were obtained from GenBank publications [ , ]. Data for SRA was obtained from publications for 2008 to 2010 [ - ] and estimated for 2007 on the basis of 44 projects being in the database at the end of the year and using February 2008 data from NCBI to estimate the approximate number of bases likely to have been submitted from that spectrum of projects. Key advances in sequencing technology are shown with arrows. The development of second generation sequencing technologies and single-molecule sequencing has had a dramatic increase in the number of sequences deposited in public databases. Less than a year after its initiation, the SRA had already surpassed classical GenBank and it now accounts for over 95% of all new sequence deposits.

Article Snippet: Pacific Biosciences has developed another sequencing-by-synthesis approach using fluorescently labeled nucleotides.

Techniques: Sequencing

Journal: Genome Biology

Article Title: The properties and applications of single-molecule DNA sequencing

doi: 10.1186/gb-2011-12-2-217

Figure Lengend Snippet: Overview of single-molecule sequencers . The three most advanced single-molecule sequencing systems all carry out sequencing-by-synthesis using laser excitation to generate a fluorescent signal from labeled nucleotides, which is then detected using a camera. (a) In the Helicos BioSciences system , single nucleotides, each with a fluorescent dye attached to the base, are sequentially added. (b,c) In the Pacific Biosciences and Life Technologies systems, four different nucleotides, each with a different color dye attached to the phosphates, are continuously added. Background fluorescence is minimized differently in the three systems. (a) Helicos uses total internal reflectance fluorescence (TIRF) to create a narrow evanescent field of light in which the intensity of the light decays exponentially away from the glass surface. Only dyes that are in the TIRF evanescent field can fluoresce. (b) Pacific Biosciences uses a zero mode waveguide (ZMW), which limits illumination to a narrow region near the bottom of the well containing the polymerase. Only dyes near the opening of the ZMW can fluoresce. (c) Life Technologies uses fluorescence resonance energy transfer (FRET) between the initially absorbing quantum dot on the polymerase and the emitting dye on the nucleotide. Only dyes close to the polymerase-attached quantum dot can be excited by FRET and then fluoresce. For the three systems, DNA is immobilized for viewing over time by a surface-attached sequencing primer (Helicos (a)), by interaction with a surface-bound polymerase (Pacific Biosciences (b)), or by ligating to a surface-attached oligonucleotide (Life Technologies (c)). For Helicos (a), the polymerase is replaced after every cycle of nucleotide addition. For Life Technologies (c), the polymerase can be replaced on a given DNA molecule after each read is completed. For Pacific Biosciences (b), the polymerase cannot be replaced.

Article Snippet: Pacific Biosciences has developed another sequencing-by-synthesis approach using fluorescently labeled nucleotides.

Techniques: Sequencing, Labeling, Fluorescence, Förster Resonance Energy Transfer