Klaus Mühlegger

Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers

We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1:05 0:33 ns (Cy5-dCTP), 2:07 0:59 ns (MR121-dUTP) and 3:88 1:71 ns (Bodipy-dUTP).

STEREOELECTRONIC EFFECTS OF MODIFIED PURINES ON THE SUGAR CONFORMATION OF NUCLEOSIDES AND FLUORESCENCE PROPERTIES

Abstract Conformational analyses of the sugar moieties of a series base-modified purine-2′-deoxynucleosides on the basis of vicinal [1H,1H] coupling constants is presented (PSEUROT 6.2) Fluorescence data of several 7-deaza- and 8-azapurine 2′-deoxynucleosides are given.

A new option in solid phase synthesis of DNA-fragments

Abstract the use of a new support suitable for enzymatic conversion of the immobilized oligonucletide chain and the possibility of post-synthesis deprotection without release from the polymer is described.

OESTRADIOL, A NEW HAPTEN FOR DETECTING NUCLEIC ACID SEQUENCES BY FISH

Abstract Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.