Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA

Augustin, Martin; Ankenbauer, W.; Angerer, Bernhard

doi:10.1016/s0168-1656(00)00420-x

Cited by 50 publications

(38 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It is also possible to use a more promiscuous polymerase (34,35) or nucleotide analogues whose dye can be chemically removed at each step. Because some of these ideas have already been used to synthesize long DNA molecules with every base replaced with nucleotide analogues (20,(34)(35)(36), we believe that there are no fundamental or practical obstacles to extending our results to create a highly parallel and sensitive single molecule sequencing technology.…”

Section: Resultsmentioning

confidence: 99%

Sequence information can be obtained from single DNA molecules

Braslavsky

Hébert

Kartalov

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

427

245

View full text Add to dashboard Cite

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.T he Sanger method of DNA sequencing (1) and subsequent developments in automation (2) and computation (3) revolutionized the world of biological sciences and eventually led to the sequencing of the consensus human genome (4, 5). The successes of this and other genome projects have only whetted the appetite of the scientific community, and many applications of DNA sequencing have been proposed that will require cheaper, faster, or more sensitive sequencing technology than conventional methods currently provide. After the determination of the consensus human genome, there is a desire to sequence many individual human genomes to provide highresolution genotypes that can be used to determine the complex relationships among disease, pharmaceutical efficacy, and genetic variability (6-8). Similarly, aggressive technological innovation is required for the field of comparative genomics to reach its full potential (4). Finally, mRNA sequencing is valuable to determine exon splicing patterns (9) and as a tool to discover gene function from context-specific expression data (10).There have been many proposals to develop new sequencing technologies based on single molecule measurements, generally either by observing the interaction of particular proteins with DNA (6, 11-13) or by using ultra high-resolution scanned probe microscopy (14). Although none of these methods has been demonstrated experimentally, they are interesting because they promise high sensitivity, low cost, and in some cases a high degree of parallelization (15). Unlike conventional technology, their speed and read length would not be inherently limited by the resolving power of electrophoretic separation. Single molecule sensitivity might permit direct sequencing of mRNA from rare cell populations or perhaps even individual cells.A major obstacle in the development of single molecule sequencing schemes is that DNA has an extraordinarily ...

show abstract

Section: Resultsmentioning

confidence: 99%

Sequence information can be obtained from single DNA molecules

Braslavsky

Hébert

Kartalov

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

427

245

View full text Add to dashboard Cite

show abstract

“…Single molecule sequencing is a goal that has been pursued for almost two decades as a possible candidate to replace the ubiquitous Sanger method (Jett et al, 1989) Different schemes have been proposed to achieve this goal, for example: (1) using exonuclease on flowstretched labeled DNA and to detect the fluorescent product down stream (Augustin et al, 2001;Werner et al, 2003), (2) stretching DNA molecules in nano fabricated devices and to read fluorescent tags at the output (Chan et al, 2004), (3) recording the ionic current through nano channels while single DNA is thread through it (Meller et al, 2000), (4) following the synthesis of DNA in real time by local confinement of illumination (Levene et al, 2003), and (5) monitoring fluorescently labeled nucleotide incorporation on single DNA molecule step by step in cycle-extensions (Braslavsky et al, 2003). From all of the above, the demonstration that sequence information can be obtained from single DNA molecules by cyclic synthesis (Braslavsky et al, 2003) lead to the development of the first working scheme for large scale single molecule sequencing (Harris et al, to be published).…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule Detection

2006

Principles of Fluorescence Spectroscopy

View full text Add to dashboard Cite

“…6,7 Certain kinds of deoxynucleotide triphosphate (dNTP) derivatives possessing a fluorescent reporter group at an appropriate position on the nucleobase can work as effective substrates for the enzymatic incorporation by polymerase and are used as fluorescent probes for the detection of the hybridization and amplification of DNA. 3,[8][9][10][11][12] Pyrene fluorophores with unique fluorescent properties, capable of generating an excited dimer with a characteristic green light, were conjugated to oligonucleotides for the structural study of the folding and development of single nucleotide polymorphisms (SNPs). In this study, we demonstrated the preparation of the deoxyuridine triphosphate derivatives labelled with the pyrene (Py) fluorophore through an ethylene glycol linker, and evaluation of the enzymatic incorporation of the triphosphate by DNA polymerase and the emission properties of the Py-incorporated DNA.…”

mentioning

confidence: 99%

Preparation of fluorescent nucleic acids generating unique emission by primer extension reaction using pyrene-labeled deoxyuridine triphosphate derivatives

Takada¹,

Tanimizu²,

Nakamura³

et al. 2014

Rapid Communication in Photoscience

View full text Add to dashboard Cite

Fluorescent nucleic acids were prepared utilizing the polymerase extension (PEX) reaction to incorporate fluorescent molecules. 2'-Deoxyuridine triphosphate (dUTP) derivatives possessing pyrene molecules as fluorophores were synthesized using the aqueous-phase Sonogashira coupling between 5-Iodo-dUTP and acetylene-linked pyrene molecules. The incorporation of the pyrene (Py)-labeled deoxyuridine triphosphates (PyU) into DNA by polymerase was evaluated by polyacrylamide gel electrophoresis, demonstrating that the PyU can work as a good substrate for the PEX reaction. The fluorescent properties of the functionalized DNA prepared by the PEX reaction were characterized by steady-state fluorescence measurements. The Py-conjugated DNA showed typical emission spectra of the pyrene, and the DNA with two pyrene molecules connected to each other by a diethylene glycol linker exhibited a broadened emission attributed to the electronic interaction between the Py molecules.Nucleic acids modified with functional groups/molecules are of significant interest as potential applications in chemical biology, diagnostic tools or nanotechnology and material science.1-3 To expand their applications as versatile tools, various kinds of oligodeoxynucleotide modified with functional molecules have been prepared by a general synthetic approach using the corresponding phosphoroamidite units during solid-phase DNA synthesis and the combination with post-synthetic oligonucleotides modification. 4,5 As an alternative approach to the conventional DNA synthesis for the preparation of the functionalized oligonucleotides, an enzymatic method using the polymerase extension (PEX) reaction has attracted much attention because of the facile preparation of longer oligonucleotides from DNA templates and multiple incorporation of functional groups/molecules into DNA based on the sequence of the templates. 6,7 Certain kinds of deoxynucleotide triphosphate (dNTP) derivatives possessing a fluorescent reporter group at an appropriate position on the nucleobase can work as effective substrates for the enzymatic incorporation by polymerase and are used as fluorescent probes for the detection of the hybridization and amplification of DNA. 3,[8][9][10][11][12] Pyrene fluorophores with unique fluorescent properties, capable of generating an excited dimer with a characteristic green light, were conjugated to oligonucleotides for the structural study of the folding and development of single nucleotide polymorphisms (SNPs). In this study, we demonstrated the preparation of the deoxyuridine triphosphate derivatives labelled with the pyrene (Py) fluorophore through an ethylene glycol linker, and evaluation of the enzymatic incorporation of the triphosphate by DNA polymerase and the emission properties of the Py-incorporated DNA.Multiple incorporation of the pyrene molecules into DNA by the polymerase extension (PEX) reaction is schematically shown in Figure 1. We have chosen pyrene (Py) as the fluorescent molecule because pyrene is known to show a strong e...

show abstract

Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA

Cited by 50 publications

References 25 publications

Sequence information can be obtained from single DNA molecules

Sequence information can be obtained from single DNA molecules

Single-Molecule Detection

Preparation of fluorescent nucleic acids generating unique emission by primer extension reaction using pyrene-labeled deoxyuridine triphosphate derivatives

Contact Info

Product

Resources

About