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

Abstract

Farbe zeigt Kernbase an: Die enzymatische templatgesteuerte Verlängerung von Primern mit fluorophormarkierten Nucleosidtriphosphaten ist die am häufigsten für die Bestimmung von DNA‐Sequenzen eingesetzte Reaktion. Nun ist die Primerverlängerung mit anschließendem optischem Auslesen ohne Enzyme möglich.

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

“…These features support the use of templated reactions in nucleic acid detection in which product formation indicates the presence of the target structure. [2,3] Several chemical reactions have been used in a DNA-or RNA-templated fashion, including organic reactions such as ligation, [4][5][6][7][8][9][10][11] extension, [12,13] cleavage, [14][15][16][17][18] and transfer [19][20][21] reactions as well as organometal approaches. [22][23][24] The work in the field of nucleic acid diagnostics addresses two major issues, selectivity and sensitivity.…”

Synthesis of 3′‐BODIPY‐Labeled Active Esters of Nucleotides and a Chemical Primer Extension Assay on Beads

“…As we have previously shown that fluorophores may also be attached to 3Ј-amines of dideoxynucleotides through a photolabile linker, [9] it is interesting to ask whether the current results also help to pave the way for multiple, stepwise primer extension reactions with optical read-out. If so, an inexpensive alternative sequencing method may have moved one step closer to medical re-sequencing of short stretches of DNA.…”

“…Labeled monomers for CPE such as 2 [8] and 3 [9] ( Figure 1) have been developed. Chemical primer extension is sensitive [10] and rapid, [11] and it allows sequence information on DNA and RNA templates to be read, [12] but known versions have been limited to assays with a purification step before detection.…”

“…monophosphate with a primer, as directed by the templating base of a DNA or RNA strand. Labeled monomers for CPE such as 2 [8] and 3 [9] (Figure 1) have been developed.…”

“…Chemical primer extension is sensitive [10] and rapid, [11] and it allows sequence information on DNA and RNA templates to be read, [12] but known versions have been limited to assays with a purification step before detection. [9] For example, in a published version of CPE with labeled monomers and read-out on DNA microarrays, labeled oligonucleotides had to be separated from excess monomers by spin columns. [9] The main cause for this undesirable feature of the assays was the background signal from unincorporated monomer on the surface of the microarrays, which was caused by unfavorable physical properties of the labeled monomers that we were unable to wash off from the surfaces of the microarray.…”