Biomedical subjects

Alexander Krieg

Publications and source records attributed to Alexander Krieg.

6 recordsLinked to original sources

PubMed2005

Towards single-molecule DNA sequencing: assays with low nonspecific adsorption.

New DNA sequencing techniques are currently being developed using single-molecule fluorescence-based detection of enzymatic double-strand synthesis. Such application requires surface architectures on which single-stranded templates can be immobilized. A further important attribute is a very low tendency to attract fluorescently labeled bases nonspecifically. On this account, the adsorption behaviour of Cy5-dNTPs on a variety of surface coatings was studied by performing real-time measurements of the DNA synthesis using a supercritical angle fluorescence biosensor. It is demonstrated that polyacrylic acid coatings are an excellent choice to minimize the nonspecific binding of the bases.

Adsorption↗

PubMed2005

DNA-intercalation on pyrene modified surface coatings.

Utilising the strong affinity between nucleic acids and an intercalating pyrene derivate, a novel efficient method for unspecific immobilisation of double-stranded DNA on to solid support for applications in bioanalytic, biophysics and microbiology is presented.

DNA↗

PubMed2005

Microscope objective for large-angle fluorescence used for rapid detection of single nucleotide polymorphisms in DNA hybridization.

A new type of microscope objective is used for the rapid detection of sequence-dependent affinity variations in DNA hybridization. We demonstrate that by performing probe/target hybridization on coverslips at room temperature terminal SNPs (single nucleotide polymorphisms) can be detected within seconds. The study of weak pair interaction, such as the association of very short DNA oligomers, requires the use of high analyte concentrations of both partners to generate a detectable amount of associated pairs. The background of high concentrations of unbound fluorescing analyte can easily hide the low signal of a weakly affine reaction and makes association extremely difficult to detect. Fluorescence detection is a powerful approach to analyze minute amounts of material, even single molecules, but it is usually limited to rather low concentrations. This limitation is now overcome due to the new type of microscope objective, which produces an extremely small detection volume at a water/glass interface.

Carbocyanines↗

PubMed2004

Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence.

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

Base Pair Mismatch↗

PubMed2004

Real-time detection of polymerase activity using supercritical angle fluorescence.

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

Biosensing Techniques↗

PubMed2003

Real-time detection of nucleotide incorporation during complementary DNA strand synthesis.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.

Base Sequence↗