Real-Time DNA Sequencing from Single Polymerase Molecules
Секвенирование ДНК в реальном времени по отдельным молекулам ДНК-полимеразы
2008-11-20
SCID: 54.1/q9puw2by
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DNA polymerasefluorescently labeled dNTPspolymerization dynamicssingle-molecule real-time sequencingzero-mode waveguide arrays
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Abstract (AI)
We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
Key Findings
1
At 15-fold coverage, consensus sequences reached 99.3% median accuracy, with no systematic errors beyond fluorophore-dependent error rates.
2
Single-molecule data revealed distinct polymerization states and pause sites associated with DNA secondary structure.
3
Single-molecule, real-time DNA sequencing was achieved by observing uninterrupted polymerase synthesis with four distinguishable fluorescent dNTPs.
4
Terminal-phosphate fluorophore labeling allowed continuous observation of DNA synthesis over thousands of bases without steric hindrance.
5
Zero-mode waveguide arrays enabled optical confinement and parallel detection of thousands of individual sequencing reactions.
Research Object
single DNA polymerase molecules performing real-time template-directed DNA synthesis
Research Subject
temporal nucleotide incorporation, polymerase dynamics, pausing at DNA secondary structure, and sequencing accuracy during uninterrupted synthesis
Publication Details
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2008-11-20
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