Directly labeled DNA probes using fluorescent nucleotides with different length linkers

Zhu, Zhengrong; Chao, Jean; Yu, Hong; Waggoner, Alan S.

doi:10.1093/nar/22.16.3418

Cited by 133 publications

(82 citation statements)

References 15 publications

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“…The yield was reduced to Ϸ10% for the second incorporation. Others have shown that nucleotide analogs with longer linker arms can be incorporated into DNA templates with significantly higher yields (33). It is also possible to use a more promiscuous polymerase (34,35) or nucleotide analogues whose dye can be chemically removed at each step.…”

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.

425

245

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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 ...

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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.

425

245

View full text Add to dashboard Cite

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“…It is known that some modified DNA polymerases are highly tolerable for nucleotides with extensive modifications with bulky groups such as energy transfer dyes at the 5-position of the pyrimidines (T and C) and 7-position of purines (G and A) (23,24). The ternary complexes of a rat DNA polymerase, a DNA templateprimer, and dideoxycytidine triphosphate have been determined (22), which supports this fact.…”

mentioning

confidence: 56%

“…However, no success has been reported for the incorporation of such a nucleotide with a cleavable fluorescent dye on the 3Ј position by DNA polymerase into a growing DNA strand. The reason is that the 3Ј position on the deoxyribose is very close to the amino acid residues in the active site of the polymerase, and the polymerase is therefore sensitive to modification in this area of the ribose ring, especially with a large fluorophore (22).It is known that some modified DNA polymerases are highly tolerable for nucleotides with extensive modifications with bulky groups such as energy transfer dyes at the 5-position of the pyrimidines (T and C) and 7-position of purines (G and A) (23,24). The ternary complexes of a rat DNA polymerase, a DNA templateprimer, and dideoxycytidine triphosphate have been determined (22), which supports this fact.…”

mentioning

confidence: 99%

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Kim

et al. 2006

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

183

160

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DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3-O-allyldNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3-O-allyl group on a DNA extension product, which is generated by incorporating 3-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pdcatalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner. DNA chip DNA sequencing is driving genomics research and discovery. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresis-based sequencing technology (2-5), a variety of new DNA-sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7-9), sequence-specific detection of single-stranded DNA using engineered nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (12), sequencing of single DNA molecules (13), and polymerase colonies (14) have been widely explored.The concept of DNA SBS was revealed in 1988 with an attempt to sequence DNA by detecting the pyrophosphate group that is generated when a nucleotide is incorporated in a DNA polymerase reaction (15). Pyrosequencing, which was developed based on this concept and an enzymatic cascade, has been explored for genome sequencing (16). However, there are inherent difficulties in this method for determining the number of incorporated nucleotides in homopolymeric regions of the template. Additionally, each of the four nucleotides needs to be added and detected separately, which increases the overall detection time. The accumulation of undegraded nucleotides and other components could also lower the accuracy of the method when sequencing a long DNA template. It is thus desirable to have a simple method t...

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“…The amount of probe used for hybridization depends on the array format and labeling method [6]. In a typical hybridization reaction, equal amounts of Cy3-labeled probes based on the incorporated dye concentration are combined [7][8][9][10][11][12][13][14][15][16].…”

Section: Resultsmentioning

confidence: 99%

AA-Dutp-Cy3 a Novel Fluorescent Labeling Agent for Nucleotides

Chaudhuri¹

2014

Biochem & Pharmacol

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A bioassay was developed to provide a process for the synthesis of cyanine dye labeled base modified nucleoside triphosphates analogues as a novel labeling agent for biomolecules. It is also to provide a low cost process and more specifically relates to a process for the synthesis of cyanine fluorophore -base modified nucleoside triphosphates analogues as well as for efficient and robust labeling of RNA and cDNA as hybridization probe in microarray detection and analysis.

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Directly labeled DNA probes using fluorescent nucleotides with different length linkers

Cited by 133 publications

References 15 publications

Sequence information can be obtained from single DNA molecules

Sequence information can be obtained from single DNA molecules

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

AA-Dutp-Cy3 a Novel Fluorescent Labeling Agent for Nucleotides

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