Four‐Color, Enzyme‐Free Interrogation of DNA Sequences with Chemically Activated, 3′‐Fluorophore‐Labeled Nucleotides

Griesang, Niels; Gießler, Kerstin; Lommel, Tanja; Richert, Clemens

doi:10.1002/anie.200600804

Cited by 29 publications

(24 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Still, the differences are not so strong as to prevent copying in less favorable cases. Chemical genotyping with fluorescent nucleotides (25) should benefit from this property of DNA.…”

Section: Discussionmentioning

confidence: 99%

“…This leaves open the question how good a template DNA is for primer extension, and thus replication. Sequence-dependent effects are important for replication and (23) for improving singlemolecule sequencing (24) and genotyping by chemical primer extension (25).…”

mentioning

confidence: 99%

“…The amino group at the primer terminus is isosteric and isoelectronic to the hydroxy group of natural DNA, and can be expected not to bias the templating efficiency. More than one extension of amino primers may be induced (25).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Templating efficiency of naked DNA

Kervio

Hochgesand

Steiner

et al. 2010

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

Self Cite

111

View full text Add to dashboard Cite

Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with ≥93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows ≥72% of the correct extension product in all cases, and ≥90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

show abstract

“…Still, the differences are not so strong as to prevent copying in less favorable cases. Chemical genotyping with fluorescent nucleotides (25) should benefit from this property of DNA.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Templating efficiency of naked DNA

Kervio

Hochgesand

Steiner

et al. 2010

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

Self Cite

111

View full text Add to dashboard Cite

show abstract

“…Recently, chemical ligation, non-enzymatic joining of DNA ends by covalent bonds, has attracted a great deal of interest, because it is free from most of the restrictions that ligases require. [5][6][7][8][9][10] This novel technique would be useful for not only biomedical but also synthetic applications such as construction of DNA-based unique nanostructures. [11][12][13] Among the various methods for templatedirected chemical ligation reported so far, photoligation has several advantages, including the lack of a need for additives, low cost, and ease of reaction control by wavelength, light strength, and irradiation time.…”

Section: Introductionmentioning

confidence: 99%

The Effect of Local Structural Disruption on the Yield of Photochemical Ligation between Anthracene-Oligonucleotide Conjugates

et al. 2008

View full text Add to dashboard Cite

The techniques of chemical ligation have attracted great attention as an alternative to enzymatic joining of DNA ends. Here we introduce the photoligation of anthracene-modified ODN conjugates through anthracene cyclodimer formation. The effect of the positions and the kinds of single base mismatch on the template was evaluated using eight templates with one-base displacements. We found out that the yield of the ligation was affected by mispairing in a positiondependent manner. Such results would be attributed to the disruption of the local structure at the ligation site.

show abstract

“…Thus far, we have focused on individual replication steps that extend the primer by a single nucleotide, and not the generation of long reads. Multiple extension steps are currently under investigation [16], but it will take some time before the results can be compiled in a meaningful fashion. Finally, in much of our work, we have included competitive reactions, where all four activated nucleotides compete in the same solution, while analyzing the product distribution by matrix-assisted laserdesorption-ionization mass spectrometry (MALDI-TOF-MS) under conditions that allow for quantitative detection [17].…”

mentioning

confidence: 99%

Chemical Primer Extension: Individual Steps of Spontaneous Replication

Stütz

Kervio²,

Deck³

et al. 2007

Chemistry & Biodiversity

Self Cite

View full text Add to dashboard Cite

The replication of genetic information, as we know it from today's biology, relies on template-directed, polymerase-catalyzed extension of primers. It is known that short stretches of complementary RNA can form on templates in the absence of enzymes. This account summarizes recent work on efficient enzyme-free primer extension, both with 3'-amino-terminal deoxyribonucleotide primers and with primers made of unmodified RNA. Near-quantitative primer extension with half-life times on the order of hours has been demonstrated by using azaoxybenzotriazolides of nucleotides and downstream-binding oligomers. Further, small non-nucleosidic substituents placed on the terminus of the template or the downstream-binding oligomer have been shown to increase the rate and fidelity of primer-extension reactions. Since all four templating bases (A, C, G, T/U) direct sequence-selective primer-extension steps, we feel that there is renewed hope that full, nonenzymatic replication from monomers may eventually be achieved.

show abstract

Four‐Color, Enzyme‐Free Interrogation of DNA Sequences with Chemically Activated, 3′‐Fluorophore‐Labeled Nucleotides

Abstract: Color reveals base: The enzymatic template‐directed elongation of primers with fluorophore‐labeled nucleoside triphosphates is the most frequently used reaction for determining DNA sequences. Primer extension followed by optical read out can now be achieved without enzymes.

Cited by 29 publications

References 36 publications

Templating efficiency of naked DNA

Templating efficiency of naked DNA

The Effect of Local Structural Disruption on the Yield of Photochemical Ligation between Anthracene-Oligonucleotide Conjugates

Chemical Primer Extension: Individual Steps of Spontaneous Replication

Contact Info

Product

Resources

About