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T Tenkanen

Publications and source records attributed to T Tenkanen.

4 recordsLinked to original sources
PubMed2006

Genome-controlled reverse transcriptase-polymerase chain reaction for targeted gene-expression analysis.

OBJECTIVE: Although gene-expression profiling has an important part to play in the classification of tumours and premalignant conditions, reproducibility of the present polymerase chain reaction (PCR)-based quantitative techniques needs to be improved for diagnostic purposes and to enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. We have developed reverse transcriptase-PCR-based technology for quantitative assessment of the relative content of multiple mRNA transcripts in small tissue or cell samples. MATERIAL AND METHODS: A multiplexed sequence modifying cDNA synthesis reaction is performed with this technique to create a 4-5 degrees increase in the melting temperature of subsequent short (56-64 bp) PCR amplicons. Each cDNA template is competitively co-amplified with genomic DNA, which serves as a universal internal standard. The relative amounts of cDNA and genomic DNA-derived amplicons are quantified in-tube by homogeneous melting curve analysis. RESULTS: The dynamic range of the assay was three orders of magnitude, while the detection limit was 100 cDNA molecules. A prototype assay, consisting of the analysis of eight genes, displayed good reproducibility (inter-assay CV 5-20 %) compared to the TaqMan assay (inter-assay CV 7-43 %). Gene-expression analysis could be performed in 20 of 20 (100 %) archival frozen samples, in 30 of 35 (86 %) archival FFPE samples and in 26 of 27 (96 %) endoscopic biopsies. CONCLUSIONS: We demonstrate that this new technique enables accurate analysis of mRNA expression in cultured cells and endoscopic tissue biopsies. Sensitive analysis FFPE tissue is also possible thanks to the short PCR amplicons.

Biopsy↗
PubMed2000

Site-specific restriction endonucleases in cyanobacteria.

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.

Anabaena↗
PubMed1991

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity.

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

Archaea↗