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Jonas Eriksson

Publications and source records attributed to Jonas Eriksson.

6 recordsLinked to original sources
PubMed2004

Improvements in Pyrosequencing technology by employing Sequenase polymerase.

Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

DNA Primers↗
PubMed2004

Pyrosequencing trade mark technology at elevated temperature.

To date, the Pyrosequencing trade mark technology has been performed at 28 degrees C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37 degrees C. By increasing the temperature to 37 degrees C, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37 degrees C enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA em leader 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.

Betaine↗
PubMed2004

7-Deaza-2'-deoxyadenosine-5'-triphosphate as an alternative nucleotide for the pyrosequencing technology.

A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7-deaza-2'-deoxyadenosine-5'-triphosphate (c7dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPalphaS). The inhibitory effect dATPalphaS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c7dATP for the dATPalphaS. Both analogs show high stability after long time storage at + 8 degrees C. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.

Apyrase↗
PubMed2003

Experimental and theoretical multiple kinetic isotope effects for an SN2 reaction. An attempt to determine transition-state structure and the ability of theoretical methods to predict experimental kinetic isotope effects.

The secondary alpha-deuterium, the secondary beta-deuterium, the chlorine leaving-group, the nucleophile secondary nitrogen, the nucleophile (12)C/(13)C carbon, and the (11)C/(14)C alpha-carbon kinetic isotope effects (KIEs) and activation parameters have been measured for the S(N)2 reaction between tetrabutylammonium cyanide and ethyl chloride in DMSO at 30 degrees C. Then, thirty-nine readily available different theoretical methods, both including and excluding solvent, were used to calculate the structure of the transition state, the activation energy, and the kinetic isotope effects for the reaction. A comparison of the experimental and theoretical results by using semiempirical, ab initio, and density functional theory methods has shown that the density functional methods are most successful in calculating the experimental isotope effects. With two exceptions, including solvent in the calculation does not improve the fit with the experimental KIEs. Finally, none of the transition states and force constants obtained from the theoretical methods was able to predict all six of the KIEs found by experiment. Moreover, none of the calculated transition structures, which are all early and loose, agree with the late (product-like) transition-state structure suggested by interpreting the experimental KIEs.

Journal Article↗
PubMed2003

Expression of the sodium channel transcripts Na(v)1.8 and Na(v)1.9 in injured dorsal root ganglion neurons of interferon-gamma or interferon-gamma receptor deficient mice.

Changes in the dorsal root ganglion (DRG) expression of the sodium channels Na(v)1.8 and Na(v)1.9 may contribute to injury-induced hyperexcitability and pain. Interferon (IFN)-gamma receptor -/- mice display a reduced pain-related behavior after nerve injury as compared to wild-type mice (NeuroReport 8 (1997) 1311). To elucidate a possible role for IFN-gamma in the regulation of sodium channels, we have studied the DRG mRNA expression of Na(v)1.8/Na(v)1.9 in IFN-gamma- or IFN-gamma receptor-deficient mice. In both types of mice, nerve damage induced a downregulation of Na(v)1.8 as well as Na(v)1.9. The magnitude of this reduction was similar to that observed in wild-type animals. These results indicate that the downregulation of Na(v)1.8/Na(v)1.9 in damaged DRG neurons is not influenced by IFN-gamma. Thus, the reduced pain-related behavior of nerve-injured IFN-gamma receptor null mice is not due to differential changes in the regulation of Na(v)1.8/Na(v)1.9 mRNA.

Animals↗