Achim E. Karger

Separating DNA sequencing fragments without a sieving matrix

The possibility of separating appropriately labeled DNA fragments using free‐flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free‐flow separation of double‒stranded DNA (dsDNA) fragments in the 100—1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end‒labeled free‒flow electrophoresis (ELFSE) can also be used to sequence single‒stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5′‒end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte‒wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera

Fluorescent and chemiluminescent detection of DNA hybrids on polymer membranes has been investigated using a cryogenically cooled, slow readout, two dimensional CCD camera in an imaging mode. The fluorescent background characteristics of commercially available nylon blotting membranes and a polypropylene membrane modified to bind DNA have been studied. The polypropylene membrane exhibits a 15-fold increase in DNA binding, 3-fold less background fluorescence and less background noise than nylon blotting membranes. However the detection limits determined from vacuum slot blots of crosslinked fluoresceinlabelled oligonucleotides show that the improved qualities of the polypropylene support do not result in a lower detection limit. This is mainly due to background noise arising from sources other than the membrane itself during the blotting/washing procedure and to a low signal-to-amount of DNA ratio with the polypropylene membrane. The lowest amount of fluorescein-labelled oligonucleotide detectable is 1.4 femtomol, with a typical exposure time of 10 minutes to image a 6x9 cm area of the membrane. The detection of chemiluminescence was done using a biotin-avidin complex in combination with an enzymatic assay. The assay was carried out after hybridization with biotin-labelled probes on vacuum slot blots with crosslinked target DNA. The detection limit is 0.12 femtomol of DNA target, a result obtained after 30 mm exposure. Further improvements are necessary to image sequencing blots with typically 1 femtomol or less of DNA per band in an acceptable exposure time.

Methylation-Dependent Fragment Separation: Novel Analysis Of 5-Methyl Cytosine By Capillary Electrophoresis Of Amplified Dna Using Pcr Incorporation Of Chemically Modified Dctp

Methylation of the cytosine (C) ring to form 5-methyl cytosine (MeC) in normally unmethylated CpG-rich regions of promoters in genes is associated with transcriptional silencing. Quantification of MeC is of current interest in findining new biomarkers for cancer. To this end, and for basic research in epigenomics, we have investigated a new method for relatively simple measurement of MeC content by capillary electrophoresis (CE). PCR amplicons for CE analysis are generated from bisulfite-converted DNA [C → uracil (U)] using fluorescently labeled primers that anneal independent of methylation status. Resultant incorporation of C vs. T at original MeC vs. C positions can lead to separate CE peaks for signal integration that is proportional to MeC content. Furthermore, these PCR products are suitable for additional methylation analyses by sequencing, single-base extension, or TaqMan®. Interestingly, PCR using α -thio-dCTP led to greater CE separations.

Precision of parameters determined by spectrophotometric measurements: Part 2. Precision of the first-order rate constant as obtained by spectrophotometric determination

Abstract Karger, A., 1990. Precision of parameters determined by spectrophotometric measurements. Part 2. Precision of the first-order rate constant as obtained by spectrophotometric determination. Chemometrics and Intelligent Laboratory Systems , 8: 217–226. The use of a method suggested by Posener for the precision estimation of nonlinear parameters is investigated by applying it to the spectrophotometric determination of the first-order rate constant. Factors which are directly proportional to the precision of the least-squares parameters of the model equations used are calculated. The factors are given as a function of the time range of the measured absorbance vs. time curve, within which the measurement values are recorded. The proposed a priori estimation of the rate constant precision is used to evaluate different experimental methods. By computer simulation the precision of four different evaluation algorithms are compared to the analytical estimate. The application to experimental data is demonstrated for the alkaline hydrolysis of acetylsalicylic acid solution.