DNA polymerase fluorescent substrates with reversible 3′-tags

Canard, Bruno; Sarfati, Robert S.

doi:10.1016/0378-1119(94)90226-7

Cited by 81 publications

(37 citation statements)

References 9 publications

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“…Efforts were also put into the development of this principle for sequence determination using labeled nucleotides (Canard and Sarfati 1994;Cheesman 1994;Metzker et al 1994;Rosenthal 1989;Tsien et al 1991). However, Metzker et al (1994) showed that the incorporation efficiency of labeled nucleotides is low, causing nonsynchronized extension, which made it difficult to sequence more than a few bases.…”

Section: Historymentioning

confidence: 99%

Pyrosequencing Sheds Light on DNA Sequencing

Ronaghi¹

2001

Genome Research

683

393

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DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.

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Section: Historymentioning

confidence: 99%

Pyrosequencing Sheds Light on DNA Sequencing

Ronaghi¹

2001

Genome Research

683

393

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“…However, the effect of a substitution at this 3' oxygen on the polymerization of nucleotides is not well documented, probably because it was anticipated that incorporation of these analogues would not occur or would lead to mere chain termination and would, thus, be of limited value for the understanding of the mechanism. During the course of synthesizing bonified 3'-modified dNTPs (15)(16)(17), we found various analogues unable to inhibit DNA synthesis. In the present report, we present evidence that DNA polymerases, including HIV-RT, exhibit catalytic versatility at the nascent 3' end of DNA by removing 3'-oxygen and 3'-nitrogen substituents.…”

mentioning

confidence: 99%

Catalytic editing properties of DNA polymerases.

Canard¹,

Cardona²,

Sarfati³

1995

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

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Enzymatic incorporation of 2',3'-dide-ixynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.

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“…The use of a dideoxynucleotide, which acts as a chain terminator in Sanger sequencing, provided the basis for the initial development of reversible blocking groups attached to the 3'end of nucleotides. 20,21 Blocking groups, such as 3'-O-allyl-2'-deoxyribonucleoside triphosphates (dNTPs) and 3'-O-azidomethyldNTPs22, have been successfully used in CRT. 3'-blocked terminators require the cleavage of two chemical bonds to remove the fluorophore from the nucleobase and restore the 3'-OH group.…”

Section: Cyclic Reversible Terminationmentioning

confidence: 99%

Rapid Advances in Nucleic Acid Technologies for Detection and Diagnostics of Pathogens

Khan

2014

JMEN

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In this area of detection and diagnostics, traditional culture-based methods are simple to use, relatively inexpensive but too time consuming for high-throughput testing and too complex for analysis of samples with a mixture of organisms and provide little clinical information regarding the pathogen such as antibiotic resistance genes, virulence factors or strain subtypes. Nucleic Acid based technologies such as polymerase chain reaction, DNA microarrays, and high throughput automated sequencing methods have advantages over these culture based technologies which provides more molecular information as discussed above. In the last 10years, there is rapid advancement in these nucleic acid based technologies and in this review article; I will describe the current state of the art in the nucleic acid technologies for detection and diagnostics and future trends and advances in the field.Keywords: nucleic acid, detection, diagnostics, polymerase chain reaction, Citation: Khan AS. Rapid advances in nucleic acid technologies for detection and diagnostics of pathogens.

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DNA polymerase fluorescent substrates with reversible 3′-tags

Cited by 81 publications

References 9 publications

Pyrosequencing Sheds Light on DNA Sequencing

Pyrosequencing Sheds Light on DNA Sequencing

Catalytic editing properties of DNA polymerases.

Rapid Advances in Nucleic Acid Technologies for Detection and Diagnostics of Pathogens

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